Food Chemistry 124 (2011) 1411–1415 Contents lists available at ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem Antifungal activity of natural and enzymatically-modified flavonoids isolated from citrus species Maria Paula Salas a,b,*, Gustavo Céliz c, Hugo Geronazzo c, Mirta Daz c, Silvia Liliana Resnik b,d a Agencia Nacional de Promoción Científica y Tecnológica (ANCYPT), Argentina b Departamento de Industrias, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Intendente Güiraldes 2160, 1428, Ciudad Autónoma de Buenos Aires, Argentina c Instituto de Investigaciones para la Industria Química (CONICET) and Facultad de Ciencias Exactas, Universidad Nacional de Salta, Avenida Bolivia 5150, Salta, Argentina d Comisión Científica de Investigaciones, Calle 526 entre 10 y 11, La Plata, Buenos Aires, Argentina article info abstract Article history: The antifungal activity of isolated flavonoids from Citrus species, such as naringin, hesperidin and neohes- Received 2 November 2009 peridin, and enzymatically-modified derivatives of these compounds, was studied on four fungi often Received in revised form 17 June 2010 found as food contaminants: Aspergillus parasiticus, Aspergillus flavus, Fusarium semitectum and Penicillium Accepted 27 July 2010 expansum. Although all the flavonoids showed antifungal activity, the intensity of this activity depended on the type of fungus and compound used. The hesperetin glucoside laurate strongly inhibited the myce- lial growth of P. expansum, while prunin decanoate was the most inhibiting flavonoid for A. flavus, A. par- Keywords: asiticus, and F. semitectum. Flavonoids The flavonoids naringin, hesperidin and neohesperidin, obtained as byproducts at low cost from the Mycelial growth Fungal growth residues of the citrus industries, present an interesting option for these industries. Citrus Ó 2010 Elsevier Ltd. All rights reserved. 1. Introduction protection against cardiovascular diseases and certain forms of cancer (Benavente-García et al., 1997; González-Molina, Domín- Flavonoids are widely distributed compounds that are found guez-Perles, Moreno, & García-Viguera, 2010; Salah et al., 1995; mainly in plants. The most common flavonoids found in Citrus sp. Tripoli, La Guardia, Giammanco, Di Majo, & Giammanco, 2007). can be classified into different groups: flavanones, flavones, flava- Several studies have assessed the antimicrobial activity of many nols and anthocyanins (Benavente-García, Castillo, Marin, Ortuño, essential oils obtained from a wide variety of plants (Demirci, Kosßar, & Del Río, 1997). In particular, flavanones are found in Citrus as Demirci, Dinç, & Basßer, 2007; Fraternale, Giamperi, & Ricci, 2003; glycosides. Rota, Herrera, Martínez, Sotomayor, & Jordán, 2007). Some articles Flavanone glycosides are abundant constituents of citrus leaves describe the antimicrobial activity and identify flavonoids, some of and fruits. The most common Citrus flavanone glycosides are hes- them found in Citrus. Among these works, we find that Arima, Ash- peridin or 30,5,7-trihydroxy-40-methoxyflavanone-7-a-L-rhamno- ida, and Danno (2002) studied the activity against Bacillus cereus syl(1?6)-b-D-glucoside, which is found in oranges, lemons and and Salmonella enteriditis, reporting that quercetin had antibacterial other citrus, naringin or 30,5,7-trihydroxy-40-methoxyflavanone- activity against these two bacteria. Another work showed that seven 7-a-L-rhamnosyl(1?6)-b-D-glucoside in grapefruits and sour pure flavonoids, including neohesperidioside, isolated from five oranges and neohesperidin or 30,5,7-trihydroxy-40-methoxyflava- moss species, proved to have antibacterial activity over some Gram none-7-a-L-rhamnosyl(1?2)-b-D-glucoside in sour oranges. This negative bacterial strains (Basile, Giordano, López-Sáez, & Cobianchi, variety of flavonoids has been obtained as a byproduct of the Citrus 1999). Rauha et al. (2000) found that the aglycone of a flavanone, industries, and it represents an interesting option for these compa- naringenin, exhibited activity against several bacteria and Proestos, nies (Ellenrieder, 2004). Boziaris, Nychas, and Komaitis (2006) showed that the extract of Flavonoids have aroused considerable interest recently because Astanea vulgaris, a plant with high concentrations of naringenin of their potential beneficial effects on human health such as antivi- and quercetin, had a high antimicrobial capacity against a strain of ral, anti-allergic, anti-inflammatory, antioxidant activities, and Listeria monocytogenes. Mandalari et al. (2007) worked with flavo- noids isolated from bergamot peel, a byproduct of the Citrus fruit * Corresponding author at: Departamento de Industrias, Facultad de Ciencias processing industry, finding that eriodictyol strongly inhibited the Exactas y Naturales, Universidad de Buenos Aires, Intendente Güiraldes 2160, 1428, growth of seven bacteria and also Saccharomyces cervisiae. Neverthe- Ciudad Autónoma de Buenos Aires, Argentina. Tel.: +54 1145763389; fax: +54 1147920781. less, few studies have examined the antifungal activity of flavonoids. E-mail address: [email protected] (M.P. Salas). One of these studies, from Wächter, Hoffmann, Furbacher, Blake, and 0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.07.100 1412 M.P. Salas et al. / Food Chemistry 124 (2011) 1411–1415 Timmermann (1999), showed that the prenylated flavanone isolated liberates 1 lmol of p-nitrophenol per minute. The a-rhamnosidase from the shrub Eysenhardita texana showed activity against the specific activity was 18.6 U mgÀ1, whereas the b-glucosidase was opportunistic pathogen Candida albicans. Two flavones from Artemi- very low (0.0074 U mgÀ1). After that, the system was kept at 4 °C sia giraldii have been reported to exhibit activity against Aspergillus for 24 h: the solid was separated by filtration, dissolved to 10 % flavus (Zheng, Tan, Yang, & Liu, 1996). Galagin, a flavonol commonly at 96 °C, and hydrolysed again at 50 °C and pH 4.0 with a new por- found in propolis samples, proved to have inhibitory activity against tion of enzyme. After a new crystallization at 4 °C, the solid was fil- five moulds (Afaloyan & Meyer, 1997). Weidenbörner and Jha (1993) tered, washed with cold water, and dried at 50 °C. P was kept in a have focused on the study of the inhibitory effect of some flavonoids dry place. During the conversion of NAR and NEO to P and HG, against fungi commonly found in food, but so far there are not many respectively, there were no other substances that could be pro- studies about the possible antifungal activity of flavonoids, espe- duced by this hydrolysis, so the transformation to these two flava- cially flavanones, isolated from Citrus. Ortuño et al. (2006) showed nones was almost completed. that some modifications in Penicillium digitatum hyphae and the The syntheses of the other flavonoid glycoside esters were car- inhibition of the spore production were caused by neohesperidin ried out by enzymatic catalysis in organic medium, using the com- and naringin. mercial enzyme lipase B of Candida antartica, immobilized in an The increasing concern of the consumer for food safety has acrylic resin NovozymÒ 435 (donation of Novozymes Latin America pushed industries to the elimination of synthetic additives and Limited, Araucaria, Parana, Brazil) and different alkyl vinyl esters their replacement by natural additives. This change is seen as a which provided the acyl group (Sigma and Fluka). The method con- benefit in the quality and safety of food (Viuda-Martos, Ruiz-Nava- sisted in placing in a hermetic reactor acetone (150 ml) as a solvent jas, Fernández-López, & Pérez-Álvareza, 2008). and molecular sieves of 4 Å (Sigma). After that, the chosen flavo- The aim of this work is to study the activity of certain natural noid glycoside (30 mM) and the vinyl ester (300 mM) were added. and enzymatically-modified flavonoids from Citrus species against The system was heated up to 50 °C before the initiation of the reac- the growth of fungi commonly found in food. tion with the enzyme. Reaction times were variable, but were com- pleted in 24–48 h, depending on the flavonoid synthesised. Each conversion was followed until the substrate was converted to al- 2. Materials and methods most 100%. All the reactions were followed by HPLC with auto injector (234 Gilson), with UV detector (118 Gilson) and using a 2.1. Flavonoid synthesis RP-8 LiChrospher 100 Merck column (25 cm length, 4 mm internal diameter and 5 lm particle size). The isocratic elution used aceto- The analysed flavonoids were naringin (NAR), prunin (P), prunin nitrile:water as mobile phase in different proportions, according to butyrate (PB), prunin decanoate (PD), prunin laurate (PL), prunin the ester analysed, and UV detection at 280 nm. In each case, a stearate (PS), hesperidin (HES), neohesperidin (NEO), hesperetin reaction product was observed that was only esterified at carbon glucoside (HG) and hesperetin glucoside laurate (HGL). Fig. 1 number 6 of the glucose. In order to purify the synthesised esters, shows the structures and known physicochemical properties of the solvent was evaporated from the reaction mixture with a ro- the different flavonoids tested. The flavonoids NAR, HES and NEO tary evaporator at 40 °C under vacuum (final pressure 14 mmHg). were obtained from immature aborted fruits of citric origin. The The residue mixture was washed several times with hexane in or- production processes consist of grinding the fruits to an average der to eliminate