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Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
Antioxidant and antifungal activities of Smilax campestris Griseb. (Smilacaceae) a
a
Marcela Isis Morais , Maria Eduarda Amaral Pinto , Sthéfane a
a
Guimarães Araújo , Ana Hortência Fonsêca Castro , Joaquim a
b
Mauricio Duarte-Almeida , Luiz Henrique Rosa , Carlos Augusto b
b
Rosa , Susana Johann & Luciana Alves Rodrigues dos Santos Lima
a
a
Campus Centro-Oeste Dona Lindu, Universidade Federal de São João Del-Rei, Rua Sebastião Gonçalves Coelho 400, Chanadour, Divinópolis, MG 35501-296, Brazil b
Departamento de Microbiologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG 31270-901, Brazil Published online: 07 Apr 2014.
To cite this article: Marcela Isis Morais, Maria Eduarda Amaral Pinto, Sthéfane Guimarães Araújo, Ana Hortência Fonsêca Castro, Joaquim Mauricio Duarte-Almeida, Luiz Henrique Rosa, Carlos Augusto Rosa, Susana Johann & Luciana Alves Rodrigues dos Santos Lima (2014): Antioxidant and antifungal activities of Smilax campestris Griseb. (Smilacaceae), Natural Product Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2014.895728 To link to this article: http://dx.doi.org/10.1080/14786419.2014.895728
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Natural Product Research, 2014 http://dx.doi.org/10.1080/14786419.2014.895728
SHORT COMMUNICATION Antioxidant and antifungal activities of Smilax campestris Griseb. (Smilacaceae) Marcela Isis Moraisa, Maria Eduarda Amaral Pintoa, Sthe´fane Guimara˜es Arau´joa, Ana Horteˆncia Fonseˆca Castroa, Joaquim Mauricio Duarte-Almeidaa, Luiz Henrique Rosab, Carlos Augusto Rosab, Susana Johannb and Luciana Alves Rodrigues dos Santos Limaa* Campus Centro-Oeste Dona Lindu, Universidade Federal de Sa˜o Joa˜o Del-Rei, Rua Sebastia˜o Gonc alves Coelho 400, Chanadour, Divino´polis, MG 35501-296, Brazil; bDepartamento de Microbiologia, Instituto de Cieˆncias Biolo´gicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG 31270-901, Brazil
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a
(Received 27 December 2013; final version received 7 February 2014) Ethanol extract and fractions obtained from aerial parts of Smilax campestris were examined in order to determine their phenolic composition, antioxidant capacity and antifungal activities. High-performance liquid chromatography coupled with DAD analysis indicated that quercetin and rutin were the main phenolic compounds present in butanol fraction and ethanol extract, respectively. The antioxidant activity assessed by the scavenging ability on 1,1-diphenyl-2-picrylhydrazyl radical was significantly more pronounced for the ethanol extract and butanol fraction than that of the commercial antioxidant 2,6-di-tert-butyl-4-methylphenol. The antifungal activity of extract and fractions was investigated by using microdilution method against five Candida and two Cryptococcus yeast strains. Ethanol extract and fractions exhibited antifungal activities against Candida albicans, Candida glabrata, Candida krusei, Candida parapsilosis, Candida tropicalis and Cryptococcus gattii. This work provides the knowledge of profile and content of flavonoids and their antioxidant and antifungal activities in the extract and fractions of aerial parts of S. campestris. Keywords: antifungal; antioxidant; flavonoids; Smilax campestris; yeast; DPPH
1. Introduction Foods of plant origin, such as teas, herbs, oil seeds, beans, fruits and vegetables, have been suggested as natural source for antioxidants (Ara & Nur 2009). Antioxidants are substances that can delay or inhibit lipid oxidation, acting as free radical scavengers, peroxide decomposers, singlet oxygen quenchers, enzyme inhibitors and synergists. In vivo, lipid oxidation may play a role in coronary heart disease, atherosclerosis, cancer and the aging process (Rufino et al. 2011). Vitamins A, C and E, carotenoids, flavonoids and other simple phenolic compounds present in natural foods are known to possess antioxidant potential (Arnao et al. 2001). This explains why the search for natural antioxidants, especially of plant origin, has greatly increased in recent years. The plants also exhibit antifungal properties (Santos et al. 2007; Johann, Cisalpino, et al. 2010, Johann, Sa´, et al. 2010), and several compounds could be responsible for this activity, such as coumarins, terpenes and flavonoids (Cowan 1999). The incidence of invasive opportunistic mycoses has increased in immunosuppressed patients, such as those who undergo organ transplant or haematopoietic stem cell or patients with cancer and AIDS (Nucci et al. 2010).
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
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Candida spp. remain the most important cause of opportunistic mycoses worldwide, and the main invasive infections due to Candida spp. are attributed to Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis and Candia krusei (Pfaller & Diekema 2007; Nucci et al. 2010). Cryptococcus neoformans is the etiologic agent of the cryptococcosis and is the causative agent in up to 30% of mortality in AIDS patients (Idnurm et al. 2005). Cryptococcus gattii causes cryptococcal infections mostly in immunocompetent hosts. Despite several effective treatment options, such mycoses are associated with high morbidity (Nucci et al. 2010). Natural products could contribute to the discovery of new and more effective antifungal drugs. The genus Smilax (Smilacaceae) contains 350 species, which are distributed widely in tropical regions of East Asia and South and North America (Sautour et al. 2005). It is well known that the rhizomes of Smilax have several pharmacological activities, such as immunomodulatory, antibacterial, antifungal, antioxidant and hepatoprotective (Ozsoy et al. 2008). Smilax campestris Griseb. is commonly known as ‘sarsaparilla blanca.’ Its roots and rhizomes have been used in folk medicine for treatment of syphilis, rheumatism and some skin diseases as well as a diaphoretic and diuretic. Tonic beverage, which is both refreshing and beneficial (calming) to the digestive system, was prepared as 10% infusion from the leaves and aerial stems of S. campestris (Rugna et al. 2003). The biological activities of this species have been demonstrated in few studies (Rugna et al. 2003; Battista et al. 2007). Some literature data pointed out antioxidant properties of rhizome (Rugna et al. 2003) and antifungal activities of leaves and rhizomes of S. campestris (Battista et al. 2007). However, until now, there have been no data about the biological activities, profile and total flavonoid contents of the aerial parts (twigs and leaves) of S. campestris. The aim of this study was to estimate total flavonoid contents and main phenolic compounds of ethanol extract and fractions obtained from aerial parts of S. campestris and also to demonstrate their antioxidant and antifungal potential.
2. Results and discussion In this work, phytochemical tests revealed the presence of alkaloids, coumarins and flavonoids in ethanol extract. Saponins were present in ethyl acetate fraction (Ac). The alkaloids and terpenes/ sterols were found in hexane (Hex) and dichloromethane (DCM) fractions. In addition, flavonoids were found to be present in the butanol (But) and hydroalcoholic (HE) fractions, and coumarins were found in the hydroalcoholic (HE) fraction. Tannins were absent in all the fractions. The total flavonoid contents and IC50 values on the 1,1-diphenyl-2-picrylhydrazyl (DPPH)scavenging activity of ethanol extract and fractions obtained from S. campestris are presented in Table S1. Ethanol extract, fractions and positive controls 2,6-di-tert-butyl-4-methylphenol (BHT) and ascorbic acid (AA) were capable of scavenging DPPH radicals in a concentrationdependent manner. Samples exhibit activities greater than the positive control BHT, at concentrations of 1 and 10 mg/mL. As can be observed from the IC50 values (Table S1), butanol fraction was found to be the most potent antioxidant, followed by ethanol extract, HE and Ac, while Hex and DCM were the least effective scavengers (exhibited the lowest antioxidant potential with highest IC50 values). Butanol fraction can be considered as the best antioxidant amongst all the extracts under study. The IC50 values determined for ethanol extract and fractions of S. campestris on the DPPH radical were statistically significant when compared with BHT and AA. The p-value for the comparison of ethanol extract and fractions of S. campestris with BHT was 0.001. The p-value for the comparison of the ethanol extract and fractions of S. campestris with AA was 0.001, except for the butanol fraction, which had a p-value . 0.05.
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The antioxidant activity of Smilax sp has been reported earlier. In a previous study on the leaves of Smilax china, ethyl acetate and butanol extracts exhibited potent antioxidant effect on various experiments (Cha & Lee 2009). The methanol, ethyl acetate and chloroform extracts obtained from roots of Smilax chinensis exhibited antioxidant activity by the DPPH method, with IC50 values of 190.14, 79.11 and 189.47 mg/mL, respectively (Mandal et al. 2008). Ethanol extract and butanol and hydroalcoholic fractions of S. campestris presented lowest IC50 values than the extracts of S. chinensis, exhibiting a greater antioxidant activity. The butanol fraction and ethanol extract had the highest total flavonoid contents: 13.79 and 13.00 mg equivalent to quercetin/mg of extract and fraction, respectively, confirming the screening for the presence of flavonoids. The total flavonoids content of Hex, DCM, Ac and HE fractions were estimated to be between 1.56 and 7.62 mg equivalent to quercetin/mg of fraction (Table S1). Phenolic compounds, such as flavonoids, possess very strong antioxidant activity and are much more effective than vitamins C and E in protecting the cells from free radical damage (Vinson et al. 1995; Wiseman et al. 1997). The butanol fraction and ethanol extract exhibited greater antioxidant activity, compared with other fractions, as well as high levels of phenolic compounds; for this reason we chose to survey the profile of its phenolic compounds by HPLC-DAD. The chromatogram of HPLCDAD butanol partition revealed few phenolic metabolites, which are shown in Figure S1. In this chromatogram obtained at 350 nm, a peak is observed between 4 and 6 minutes, well pronounced with a similar spectrum of quercetin derivative (254, 268sh, 361). The ethanol extract displayed a similar profile (Figure S1). However, the peak with highest abundance detected was the rutin derivative (255, 262sh, 358). The results indicated that the butanol fraction, whose chromatogram highlighted the presence of derivatives of quercetin, exhibited higher antioxidant activity compared with ethanol extract at similar concentrations. This may be due to the five reactive phenolic hydroxyl groups of quercetin, which are linked to ring B (a ring which has higher reductase activity) whose aromatic nucleus provides stability to the flavonoid radical. Rutin has a structure similar to that of quercetin, but with substitution on one of the glycosidic hydroxyl groups, it therefore exhibits lower antioxidant activity (Duarte-Almeida et al. 2006). The antifungal activities of ethanol extract and fractions of S. campestris were evaluated against seven yeast strains of clinical interest (Table S2). The ethyl acetate fraction exhibited the highest activity against C. krusei, with a minimal inhibitory concentration (MIC) value of 62.5 mg/mL, and was also active against the C. gattii, with an MIC value of 1000 mg/mL. The Hex fraction exhibited activity against C. glabrata and C. krusei, with MIC values of 125 and 500 mg/mL, respectively. The ethanol extract was active against C. krusei, C. parapsilosis and C. gattii with an MIC value of 1000 mg/mL, and C. albicans, C. glabrata and C. tropicalis with an MIC value of 2000 mg/mL. The DCM fraction displayed activity against C. krusei and C. tropicalis, with an MIC value of 2000 mg/mL. Meanwhile, the butanol and hydroalcoholic fractions were inactive against all the yeast strains studied. It should be noted that the ethanol extract and fractions of S. campestris were less potent than positive controls amphotericin B and fluconazole. The MICs determined for ethanol extract and fractions of S. campestris against the Candida and Cryptococcus yeast were statistically significant when compared with fluconazole and amphotericin B. The p-value for the comparison of ethanol extract and fractions of S. campestris with fluconazole and amphotericin B was 0.001. Ethanol extract and fractions of S. campestris demonstrated antifungal activities against six yeast strains of clinical interest, C. albicans, C. glabrata, C. krusei, C. parapsilosis, C. tropicalis and C. gattii. To the best of our knowledge, this study shows for the first time the antifungal activity of ethanol extract and fractions of aerial parts of S. campestris against C. glabrata, C. krusei, C. parapsilosis and C. gattii. Antifungal activities observed in this work can be
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confirmed by other studies on S. campestris extracts that are used against C. albicans. Methanol extracts obtained from the leaves and rhizomes of S. campestris presented activity against C. albicans; however, methanol extracts of the rhizomes were more potent than that of the leaves (Battista et al. 2007). The screening for the ethyl acetate fraction suggested the presence of saponins. Saponins isolated from Smilax medica and Smilax aspera displayed inhibitory activity against Candida spp. (Sautour et al. 2005; Belhouchet et al. 2008). Thus, the antifungal activity found in the ethyl acetate fraction against C. krusei can be attributed, at least partially, to the action of saponins. The effect against Candida species is interesting. The extract and fractions tested exhibited better activity against C. krusei. C. krusei presents intrinsic resistance to fluconazole (Abbas et al. 2000). Therefore, the use of other antifungal drugs in the treatment of patient infected with this yeast is recommended. Some isolates of C. krusei are also resistant to other azoles, such as itraconazole (Mulu et al. 2013). Diseases caused by C. krusei have high associated mortality (30 – 60%) (Mun˜oz et al. 2005). The development of resistance to fluconazole among clinical strains of C. albicans, C. glabrata, C. parapsilosis and other Candida species also hampers the treatment of these infections (Bopp et al. 2006). The activity of ethanol extracts and ethyl acetate fractions against C. gattii is of particular importance, as this yeast has been implicated in HIVassociated infections in California, Botswana and Malawi (Chatuverdi et al. 2005). Amphotericin B displayed significant MIC value against these yeasts, but this drug is very toxic and its use is recommended only for hospitalised patients. Therefore, in spite of higher MIC values of the extracts from S. campestris compared with control drugs, the inhibitory activity of the extracts is interesting, the ethyl acetate extract, in particular. 3. Conclusion In this study, ethanol extract and butanol fraction of S. campestris displayed more pronounced antioxidant potential than the commercial antioxidant BHT. Quercetin and rutin were the main phenolic compounds present in butanol fraction and ethanol extract, respectively. Ethanol extract and fractions exhibited antifungal activities against C. albicans, C. glabrata, C. krusei, C. parapsilosis, C. tropicalis and C. gattii. This is the first time, to the best of our knowledge, that the profile and content flavonoids in the extract and fractions and antioxidant and antifungal activities of aerial parts of S. campestris are demonstrated. These results encourage additional studies to evaluate the possibilities of using extract and fractions of aerial parts of S. campestris as natural sources for antioxidant and antifungal activities of plant origin. Supplementary material Experimental details relating to this article is available online, alongside Tables S1 and S2 and Figure S1. Acknowledgements We thank Prof. Dr Douglas Antoˆnio de Carvalho for botanical identification of this plant.
References Abbas J, Bodey GP, Hanna HA, Mardani M, Girgawy E, Abi-Said D, Whimbey E, Hachem R, Raad I. 2000. Candida krusei Fungemia. An escalating serious infection in immunocompromised patients. Arch Intern Med. 160:2659–2664. Ara N, Nur H. 2009. In vitro antioxidant activity of methanolic leaves and flowers extracts of Lippia alba. Res J Med Med Sci. 4:107– 110.
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Arnao MB, Cano A, Acosta M. 2001. The hydrophilic and lipophilic contribution to total antioxidant activity. Food Chem. 73:239–244. Battista SM, Garcia GF, Rugna AZ, Wagner ML, Gurni AA. 2007. Actividad antimico´tica en extractos de diferentes organos de Smilax campestris Griseb-Smilacaceae. Bol Latinoam Caribe Plant Med Aromat. 6:330–331. Belhouchet Z, Sautour M, Miyamoto T, Lacaille-Dubois MA. 2008. Steroidal saponins from the roots of Smilax aspera subsp. Mauritanica. Chem Pharm Bull. 56:1324–1327. Bopp LH, Baltch AL, Ritz WJ, Michelsen PB, Smith RP. 2006. Antifungal effect of voriconazole on intracellular Candida glabrata, Candida krusei and Candida parapsilosis in human monocyte-derived macrophages. J Med Microbiol. 55:865–870. Cha BC, Lee EH. 2009. Antioxidant activities of flavonoids from the leaves of Smilax china Linne. Korean J Pharmacog. 38:31–36. Chatuverdi S, Dyavaiah M, Larsen RA, Chatuverdi V. 2005. Cryptococcus gattii in AIDS patients, southern California. Emerg Infect Dis. 11:1686– 1692. Cowan MM. 1999. Plant products as antimicrobial agents. Clin Microbiol Rev. 12:564–582. Duarte-Almeida JM, Santos RJ, Genovese MI, Lajolo FM. 2006. Avaliac a˜o da atividade antioxidante utilizando sistema b-caroteno/a´cido linole´ico e me´todo de sequ¨estro de radicais DPPH. Cieˆnc Tecnol Aliment. 26:446–452. Idnurm A, Yong-Sun B, Nielsen K, Lin X, Fraser JA, Heitman JA. 2005. Deciphering the model pathogenic fungus Cryptococcus neoformans. Nat Rev Microbiol. 3:753–764. Johann S, Cisalpino PS, Watanabe GA, Cota BB, de Siqueira EP, Pizzolatti MG, Zani CL, de Resende MA. 2010. Antifungal activity of extracts of some plants used in the Brazilian traditional medicine against the pathogenic fungus Paracoccidioides brasiliensis. Pharm Biol. 48:388–396. Johann S, Sa´ NP, Lima LARS, Cisalpino PS, Cota BB, Alves TM, Siqueira EP, Zani CL. 2010. Antifungal activity of schinol and a new biphenyl compound isolated from Schinus terebinthifolius against the pathogenic fungus Paracoccidioides brasiliensis. Ann Clin Microbiol Antimicrob. 9:30–36. Mandal SB, Jana GK, Das S, Sahu R, Venkidesh R, Dewanjee S. 2008. Hepatoprotective and antioxidant activities of Smilax chinensis L. root. Pharmacologyonline. 2:529–535. Mulu A, Kassu A, Anagaw B, Moges B, Gelaw A, Alemayehu M, Belyhun Y, Biadglegne F, Hurissa Z, Moges F, Isogai E. 2013. Frequent detection of ‘azole’ resistant Candida species among late presenting AIDS patients in northwest Ethiopia. BMC Infect Dis. 13:82. Mun˜oz P, Sa´nchez-Somolinos M, Alcala´ L, Rodrı´guez-Cre´ixems M, Pela´ez T, Bouza E. 2005. Candida krusei fungaemia: antifungal susceptibility and clinical presentation of an uncommon entity during 15 years in a single general hospital. J Antimicrob Chemother. 55:188–193. Nucci M, Queiroz-Telles F, Tobo AM, Restrepo A, Colombo AL. 2010. Epidemiology of opportunistic fungal infections in Latin America. Clin Infect Dis. 51:561–570. Ozsoy N, Can A, Yanardagb R, Akeva N. 2008. Antioxidant activity of Smilax excelsa L. leaf extracts. Food Chem. 110:571–583. Pfaller MA, Diekema DJ. 2007. Epidemiology of invasive candidiasis: a persistent public health problem. Clin Microbiol Rev. 20:133–163. Rufino MSM, Alves RE, Fernandes FAN, Brito ES. 2011. Free radical scavenging behavior of ten exotic tropical fruits extracts. Food Res Int. 44:2072–2075. Rugna A, Polo J, Evelson P, Gurni AA, Llesuy S, Wagner ML. 2003. Antioxidant activity in rhizomes from Smilax campestris Griseb. Smilacaceae. Mol Med Chem. 1:21–25. Santos GD, Ferri PH, Santos SC, Bao SN, Soares CM, Pereira M. 2007. Oenothein B inhibits the expression of PbFKS1 transcript and induces morphological changes in Paracoccidioides brasiliensis. Med Mycol. 45:609–618. Sautour M, Miyamoto T, Lacaille-Dubois MA. 2005. Steroidal saponins from Smilax medica and their antifungal activity. J Nat Prod. 68:1489–1493. Vinson JA, Dabbagh YA, Serry MM, Jang J. 1995. Plant flavonoids, especially tea flavonoids, are powerful antioxidants using an in vitro oxidation model for heart disease. J Agric Food Chem. 43:2800–2802. Wiseman SA, Balentine DA, Frei B. 1997. Antioxidants in tea. Crit Rev Food Sci Nutr. 37:705–718.
Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
Antioxidant and antifungal activities of Smilax campestris Griseb. (Smilacaceae) a
a
Marcela Isis Morais , Maria Eduarda Amaral Pinto , Sthéfane a
a
Guimarães Araújo , Ana Hortência Fonsêca Castro , Joaquim a
b
Mauricio Duarte-Almeida , Luiz Henrique Rosa , Carlos Augusto b
b
Rosa , Susana Johann & Luciana Alves Rodrigues dos Santos Lima
a
a
Campus Centro-Oeste Dona Lindu, Universidade Federal de São João Del-Rei, Rua Sebastião Gonçalves Coelho 400, Chanadour, Divinópolis, MG 35501-296, Brazil b
Departamento de Microbiologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG 31270-901, Brazil Published online: 07 Apr 2014.
To cite this article: Marcela Isis Morais, Maria Eduarda Amaral Pinto, Sthéfane Guimarães Araújo, Ana Hortência Fonsêca Castro, Joaquim Mauricio Duarte-Almeida, Luiz Henrique Rosa, Carlos Augusto Rosa, Susana Johann & Luciana Alves Rodrigues dos Santos Lima (2014): Antioxidant and antifungal activities of Smilax campestris Griseb. (Smilacaceae), Natural Product Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2014.895728 To link to this article: http://dx.doi.org/10.1080/14786419.2014.895728
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever
or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content.
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This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Natural Product Research, 2014 http://dx.doi.org/10.1080/14786419.2014.895728
SHORT COMMUNICATION Antioxidant and antifungal activities of Smilax campestris Griseb. (Smilacaceae) Marcela Isis Moraisa, Maria Eduarda Amaral Pintoa, Sthe´fane Guimara˜es Arau´joa, Ana Horteˆncia Fonseˆca Castroa, Joaquim Mauricio Duarte-Almeidaa, Luiz Henrique Rosab, Carlos Augusto Rosab, Susana Johannb and Luciana Alves Rodrigues dos Santos Limaa* Campus Centro-Oeste Dona Lindu, Universidade Federal de Sa˜o Joa˜o Del-Rei, Rua Sebastia˜o Gonc alves Coelho 400, Chanadour, Divino´polis, MG 35501-296, Brazil; bDepartamento de Microbiologia, Instituto de Cieˆncias Biolo´gicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG 31270-901, Brazil
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a
(Received 27 December 2013; final version received 7 February 2014) Ethanol extract and fractions obtained from aerial parts of Smilax campestris were examined in order to determine their phenolic composition, antioxidant capacity and antifungal activities. High-performance liquid chromatography coupled with DAD analysis indicated that quercetin and rutin were the main phenolic compounds present in butanol fraction and ethanol extract, respectively. The antioxidant activity assessed by the scavenging ability on 1,1-diphenyl-2-picrylhydrazyl radical was significantly more pronounced for the ethanol extract and butanol fraction than that of the commercial antioxidant 2,6-di-tert-butyl-4-methylphenol. The antifungal activity of extract and fractions was investigated by using microdilution method against five Candida and two Cryptococcus yeast strains. Ethanol extract and fractions exhibited antifungal activities against Candida albicans, Candida glabrata, Candida krusei, Candida parapsilosis, Candida tropicalis and Cryptococcus gattii. This work provides the knowledge of profile and content of flavonoids and their antioxidant and antifungal activities in the extract and fractions of aerial parts of S. campestris. Keywords: antifungal; antioxidant; flavonoids; Smilax campestris; yeast; DPPH
1. Introduction Foods of plant origin, such as teas, herbs, oil seeds, beans, fruits and vegetables, have been suggested as natural source for antioxidants (Ara & Nur 2009). Antioxidants are substances that can delay or inhibit lipid oxidation, acting as free radical scavengers, peroxide decomposers, singlet oxygen quenchers, enzyme inhibitors and synergists. In vivo, lipid oxidation may play a role in coronary heart disease, atherosclerosis, cancer and the aging process (Rufino et al. 2011). Vitamins A, C and E, carotenoids, flavonoids and other simple phenolic compounds present in natural foods are known to possess antioxidant potential (Arnao et al. 2001). This explains why the search for natural antioxidants, especially of plant origin, has greatly increased in recent years. The plants also exhibit antifungal properties (Santos et al. 2007; Johann, Cisalpino, et al. 2010, Johann, Sa´, et al. 2010), and several compounds could be responsible for this activity, such as coumarins, terpenes and flavonoids (Cowan 1999). The incidence of invasive opportunistic mycoses has increased in immunosuppressed patients, such as those who undergo organ transplant or haematopoietic stem cell or patients with cancer and AIDS (Nucci et al. 2010).
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
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Candida spp. remain the most important cause of opportunistic mycoses worldwide, and the main invasive infections due to Candida spp. are attributed to Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis and Candia krusei (Pfaller & Diekema 2007; Nucci et al. 2010). Cryptococcus neoformans is the etiologic agent of the cryptococcosis and is the causative agent in up to 30% of mortality in AIDS patients (Idnurm et al. 2005). Cryptococcus gattii causes cryptococcal infections mostly in immunocompetent hosts. Despite several effective treatment options, such mycoses are associated with high morbidity (Nucci et al. 2010). Natural products could contribute to the discovery of new and more effective antifungal drugs. The genus Smilax (Smilacaceae) contains 350 species, which are distributed widely in tropical regions of East Asia and South and North America (Sautour et al. 2005). It is well known that the rhizomes of Smilax have several pharmacological activities, such as immunomodulatory, antibacterial, antifungal, antioxidant and hepatoprotective (Ozsoy et al. 2008). Smilax campestris Griseb. is commonly known as ‘sarsaparilla blanca.’ Its roots and rhizomes have been used in folk medicine for treatment of syphilis, rheumatism and some skin diseases as well as a diaphoretic and diuretic. Tonic beverage, which is both refreshing and beneficial (calming) to the digestive system, was prepared as 10% infusion from the leaves and aerial stems of S. campestris (Rugna et al. 2003). The biological activities of this species have been demonstrated in few studies (Rugna et al. 2003; Battista et al. 2007). Some literature data pointed out antioxidant properties of rhizome (Rugna et al. 2003) and antifungal activities of leaves and rhizomes of S. campestris (Battista et al. 2007). However, until now, there have been no data about the biological activities, profile and total flavonoid contents of the aerial parts (twigs and leaves) of S. campestris. The aim of this study was to estimate total flavonoid contents and main phenolic compounds of ethanol extract and fractions obtained from aerial parts of S. campestris and also to demonstrate their antioxidant and antifungal potential.
2. Results and discussion In this work, phytochemical tests revealed the presence of alkaloids, coumarins and flavonoids in ethanol extract. Saponins were present in ethyl acetate fraction (Ac). The alkaloids and terpenes/ sterols were found in hexane (Hex) and dichloromethane (DCM) fractions. In addition, flavonoids were found to be present in the butanol (But) and hydroalcoholic (HE) fractions, and coumarins were found in the hydroalcoholic (HE) fraction. Tannins were absent in all the fractions. The total flavonoid contents and IC50 values on the 1,1-diphenyl-2-picrylhydrazyl (DPPH)scavenging activity of ethanol extract and fractions obtained from S. campestris are presented in Table S1. Ethanol extract, fractions and positive controls 2,6-di-tert-butyl-4-methylphenol (BHT) and ascorbic acid (AA) were capable of scavenging DPPH radicals in a concentrationdependent manner. Samples exhibit activities greater than the positive control BHT, at concentrations of 1 and 10 mg/mL. As can be observed from the IC50 values (Table S1), butanol fraction was found to be the most potent antioxidant, followed by ethanol extract, HE and Ac, while Hex and DCM were the least effective scavengers (exhibited the lowest antioxidant potential with highest IC50 values). Butanol fraction can be considered as the best antioxidant amongst all the extracts under study. The IC50 values determined for ethanol extract and fractions of S. campestris on the DPPH radical were statistically significant when compared with BHT and AA. The p-value for the comparison of ethanol extract and fractions of S. campestris with BHT was 0.001. The p-value for the comparison of the ethanol extract and fractions of S. campestris with AA was 0.001, except for the butanol fraction, which had a p-value . 0.05.
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The antioxidant activity of Smilax sp has been reported earlier. In a previous study on the leaves of Smilax china, ethyl acetate and butanol extracts exhibited potent antioxidant effect on various experiments (Cha & Lee 2009). The methanol, ethyl acetate and chloroform extracts obtained from roots of Smilax chinensis exhibited antioxidant activity by the DPPH method, with IC50 values of 190.14, 79.11 and 189.47 mg/mL, respectively (Mandal et al. 2008). Ethanol extract and butanol and hydroalcoholic fractions of S. campestris presented lowest IC50 values than the extracts of S. chinensis, exhibiting a greater antioxidant activity. The butanol fraction and ethanol extract had the highest total flavonoid contents: 13.79 and 13.00 mg equivalent to quercetin/mg of extract and fraction, respectively, confirming the screening for the presence of flavonoids. The total flavonoids content of Hex, DCM, Ac and HE fractions were estimated to be between 1.56 and 7.62 mg equivalent to quercetin/mg of fraction (Table S1). Phenolic compounds, such as flavonoids, possess very strong antioxidant activity and are much more effective than vitamins C and E in protecting the cells from free radical damage (Vinson et al. 1995; Wiseman et al. 1997). The butanol fraction and ethanol extract exhibited greater antioxidant activity, compared with other fractions, as well as high levels of phenolic compounds; for this reason we chose to survey the profile of its phenolic compounds by HPLC-DAD. The chromatogram of HPLCDAD butanol partition revealed few phenolic metabolites, which are shown in Figure S1. In this chromatogram obtained at 350 nm, a peak is observed between 4 and 6 minutes, well pronounced with a similar spectrum of quercetin derivative (254, 268sh, 361). The ethanol extract displayed a similar profile (Figure S1). However, the peak with highest abundance detected was the rutin derivative (255, 262sh, 358). The results indicated that the butanol fraction, whose chromatogram highlighted the presence of derivatives of quercetin, exhibited higher antioxidant activity compared with ethanol extract at similar concentrations. This may be due to the five reactive phenolic hydroxyl groups of quercetin, which are linked to ring B (a ring which has higher reductase activity) whose aromatic nucleus provides stability to the flavonoid radical. Rutin has a structure similar to that of quercetin, but with substitution on one of the glycosidic hydroxyl groups, it therefore exhibits lower antioxidant activity (Duarte-Almeida et al. 2006). The antifungal activities of ethanol extract and fractions of S. campestris were evaluated against seven yeast strains of clinical interest (Table S2). The ethyl acetate fraction exhibited the highest activity against C. krusei, with a minimal inhibitory concentration (MIC) value of 62.5 mg/mL, and was also active against the C. gattii, with an MIC value of 1000 mg/mL. The Hex fraction exhibited activity against C. glabrata and C. krusei, with MIC values of 125 and 500 mg/mL, respectively. The ethanol extract was active against C. krusei, C. parapsilosis and C. gattii with an MIC value of 1000 mg/mL, and C. albicans, C. glabrata and C. tropicalis with an MIC value of 2000 mg/mL. The DCM fraction displayed activity against C. krusei and C. tropicalis, with an MIC value of 2000 mg/mL. Meanwhile, the butanol and hydroalcoholic fractions were inactive against all the yeast strains studied. It should be noted that the ethanol extract and fractions of S. campestris were less potent than positive controls amphotericin B and fluconazole. The MICs determined for ethanol extract and fractions of S. campestris against the Candida and Cryptococcus yeast were statistically significant when compared with fluconazole and amphotericin B. The p-value for the comparison of ethanol extract and fractions of S. campestris with fluconazole and amphotericin B was 0.001. Ethanol extract and fractions of S. campestris demonstrated antifungal activities against six yeast strains of clinical interest, C. albicans, C. glabrata, C. krusei, C. parapsilosis, C. tropicalis and C. gattii. To the best of our knowledge, this study shows for the first time the antifungal activity of ethanol extract and fractions of aerial parts of S. campestris against C. glabrata, C. krusei, C. parapsilosis and C. gattii. Antifungal activities observed in this work can be
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confirmed by other studies on S. campestris extracts that are used against C. albicans. Methanol extracts obtained from the leaves and rhizomes of S. campestris presented activity against C. albicans; however, methanol extracts of the rhizomes were more potent than that of the leaves (Battista et al. 2007). The screening for the ethyl acetate fraction suggested the presence of saponins. Saponins isolated from Smilax medica and Smilax aspera displayed inhibitory activity against Candida spp. (Sautour et al. 2005; Belhouchet et al. 2008). Thus, the antifungal activity found in the ethyl acetate fraction against C. krusei can be attributed, at least partially, to the action of saponins. The effect against Candida species is interesting. The extract and fractions tested exhibited better activity against C. krusei. C. krusei presents intrinsic resistance to fluconazole (Abbas et al. 2000). Therefore, the use of other antifungal drugs in the treatment of patient infected with this yeast is recommended. Some isolates of C. krusei are also resistant to other azoles, such as itraconazole (Mulu et al. 2013). Diseases caused by C. krusei have high associated mortality (30 – 60%) (Mun˜oz et al. 2005). The development of resistance to fluconazole among clinical strains of C. albicans, C. glabrata, C. parapsilosis and other Candida species also hampers the treatment of these infections (Bopp et al. 2006). The activity of ethanol extracts and ethyl acetate fractions against C. gattii is of particular importance, as this yeast has been implicated in HIVassociated infections in California, Botswana and Malawi (Chatuverdi et al. 2005). Amphotericin B displayed significant MIC value against these yeasts, but this drug is very toxic and its use is recommended only for hospitalised patients. Therefore, in spite of higher MIC values of the extracts from S. campestris compared with control drugs, the inhibitory activity of the extracts is interesting, the ethyl acetate extract, in particular. 3. Conclusion In this study, ethanol extract and butanol fraction of S. campestris displayed more pronounced antioxidant potential than the commercial antioxidant BHT. Quercetin and rutin were the main phenolic compounds present in butanol fraction and ethanol extract, respectively. Ethanol extract and fractions exhibited antifungal activities against C. albicans, C. glabrata, C. krusei, C. parapsilosis, C. tropicalis and C. gattii. This is the first time, to the best of our knowledge, that the profile and content flavonoids in the extract and fractions and antioxidant and antifungal activities of aerial parts of S. campestris are demonstrated. These results encourage additional studies to evaluate the possibilities of using extract and fractions of aerial parts of S. campestris as natural sources for antioxidant and antifungal activities of plant origin. Supplementary material Experimental details relating to this article is available online, alongside Tables S1 and S2 and Figure S1. Acknowledgements We thank Prof. Dr Douglas Antoˆnio de Carvalho for botanical identification of this plant.
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