Hindawi Publishing Corporation BioMed Research International Volume 2014, Article ID 127021, 11 pages http://dx.doi.org/10.1155/2014/127021 Research Article Evaluation of Anti-Candida Activity of Vitis vinifera L. Seed Extracts Obtained from Wine and Table Cultivars Giovanna Simonetti,1 Anna Rita Santamaria,2 Felicia Diodata D’Auria,1 Nadia Mulinacci,3 Marzia Innocenti,3 Francesca Cecchini,4 Eva Pericolini,5 Elena Gabrielli,5 Simona Panella,1 Donato Antonacci,6 Anna Teresa Palamara,1 Anna Vecchiarelli,5 and Gabriella Pasqua2 1 Department of Public Health and Infectious Diseases, Sapienza University of Rome, Piazzale Aldo Moro 5, 00185 Rome, Italy 2 Department of Environmental Biology, Sapienza University of Rome, Piazzale Aldo Moro 5, 00185 Rome, Italy 3 Department of NEUROFARBA, Section of Pharmaceutical and Nutraceutical Sciences and Multidisciplinary Centre of Research on Food Sciences, Florence University, Via Ugo Schiff 6, Sesto Fiorentino, 50019 Florence, Italy 4 CRA Agricultural Research Council, Research Unit for Enology in Central Italy, Via Cantina 12, Sperimentale 1, Velletri, 00049 Rome, Italy 5 Department of Experimental Medicine, MicrobiologySection,UniversityofPerugia,ViaGambuli, Polo Unico Sant’Andrea delle Fratte, 06132 Perugia, Italy 6 CRA Agricultural Research Council, Research Unit for Table Grapes and Wine Growing in Mediterranean Environment, Via Casamassima 148, Turi, 70010 Bari, Italy Correspondence should be addressed to Gabriella Pasqua; [email protected] Received 5 December 2013; Accepted 27 March 2014; Published 23 April 2014 Academic Editor: Aramati B. Reddy Copyright © 2014 Giovanna Simonetti et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. For the first time, grape seed extracts (GSEs), obtained from wine and table cultivars of Vitis vinifera L., cultured in experimental fields of Lazio and Puglia regions of Italy and grown in different agronomic conditions, have been tested on43 Candida species strains. We demonstrated a significant correlation between the content of the flavan-3-ols in GSEs extracts, with a polymerization degree ≥4, and anti-Candida activity. Moreover, we demonstrated that GSEs, obtained from plants cultured with reduced irrigation, showed a content of polymeric flavan-3-ols >250 mg/g with geometric mean MIC values between 5.7 and 20.2 mg/L against Candida albicans reference strains. GSE, showing 573 mg/g of polymeric flavan-3-ols, has been tested in an experimental murine model of vaginal candidiasis by using noninvasive in vivo imaging technique. The results pointed out a significant inhibition of Candida albicans load 5 days after challenge. These findings indicate that GSEs with high content of polymeric flavan-3-ols can beusedin mucosal infection as vaginal candidiasis. 1. Introduction Azoles are the most common antifungal agents available to treat topical Candida infections. However, these antifungal Candida species are major human opportunistic fungal drugs have several defects related to clinical usage, such as pathogens that cause both mucosal and deep tissue infections. low efficacy and side effects. Therefore, there is an urgent need The frequency of mucosal and cutaneous fungal infections of new antifungal agents [3]. Natural anti-infective agents has dramatically increased worldwide. Infection caused by represent a promising approach for the treatment of Candida Candida spp. affects 70–75% of women at least once during infections [4]. Phytomedicine, which has historically been an their life. Recurrent vulvovaginal candidiasis occurs in 5% of important aspect of traditional medicine in nonindustrialized women with Candida vaginitis [1, 2]. Most of these infections countries, is now becoming an integral part of healthcare in are caused by Candida albicans (C. albicans) and among non- industrialized countries. Plants are the source of thousands of albicans Candida spp., C. glabrata, C. tropicalis, and C. krusei. new phytochemicals, and different strategies can be applied to 2 BioMed Research International improvetheyieldsofbioactivemetabolitesintheplantandto in 2008, and in 2011 has been 223 mm, 423 mm, and 245 mm, obtain chemically standardized extracts [5, 6]. Vitis vinifera respectively. The cultivars had training system to Cordon L. is the most important fruit species in the world, cultivated Spur, with plant density of 2.60 × 1 m. In the examined years especially in Mediterranean area. As reported by the wide the same cultural practices were applied in the vineyard. All literature [7],grapesarerichsourceofpolyphenols,impor- the grapes were harvested at technological maturation and ∘ tant secondary metabolites produced by higher plants, which frozen at −20 C. The seeds have been isolated immediately play multiple essential roles in plant physiology and showed before use and subjected to extraction process. healthy properties in human organism, mainly as antioxidant, antiallergic, anti-inflammatory, anticancer, antihypertensive, 2.2. Sample Preparation. The seeds were separated from the renoprotective, and antimicrobial agents [8, 9]. GSEs are flesh and the skin, weighed, and put in liquid nitrogen ina recognized as a complex mixture of monomeric, oligomeric, porcelain mortar and ground to obtain a fine powder. They and polymeric flavan-3-ols. The principal monomers identi- were extracted three times (24 hrs for each extraction) by the − − fied are (+)-catechin, ( )-epicatechin, ( )-epicatechin gallate mixture EtOH/H2O (7 : 3 v/v) acidified with formic acid to − − (ECg), ( )-epigallocatechin (EGC), and ( )-epigallocatechin pH 3; the ratio matrix/solvent was 1 g fresh weight/10 mL. gallate (EGCg). Several fungi, including C. albicans,are After the removal of the solid residue, the extracts were dried ∘ sensitivetoEGCg,themaincomponentofgreenteaextracts (t ≤ 30 C), weighed, and redissolved in a suitable volume of [10]. The content of flavan-3-ols in seed grapes is influenced the same extraction solution to obtain enriched extracts. The by several factors mainly cultivar, irrigation, nitrogen fertil- samples were centrifuged (12.000 rpm for 5 min) to obtain a ization, delayed harvest, and storage conditions [11]. limpid solution for the HPLC/DAD/MS analyses. Only the Moreover, the application of an extraction process suit- seeds of Abbuoto and Verdicchio cultivars (vintages 2006 and able to efficiently recover the target metabolites and an 2008) were treated with a different method using a buffer appropriate analytical method for an accurate qualitative at pH 3.2 as extractive solution. This method was applied and quantitative determination of extract components are with the aim to simulate the wine-making process. The seeds required. were manually separated from the berries and extracted with ∘ In this work, for the first time, anti-Candida activity and 125 mL of the buffer solution for 144 hours at 30 C. The buffer chemical analysis of GSEs obtained from wine and table composition consisted of tartaric acid 5 g; NaOH 1 N 22 mL; cultivars of Vitis vinifera L. grown in different agronomic Na2S2O5 2 g; and EtOH 95% 120 mL. conditions have been evaluated and compared with respect to their phenolic content. The HPLC method with a Poroshell 2.3. HPLC/DAD/MS Analysis. The multistep elution method column has allowed to quantify not only the flavan-3-ols was applied: it started with 95% H2Ofor5min,thenwith oligomers but also the polymeric forms (polymerization 86% H2Ofor25min,84%H2Ofor5min;82%H2Ofor2min, > degree 4) difficult to be detected with conventional reverse 80% H2O for 3 min and a plateau for 4 min, 70% H2Ofor phase columns. Moreover, the effect of GSE treatment on 3 min and a plateau for 3 min, up to 20% H2Ofor4minanda an experimental murine model of vaginal candidiasis was plateau for 5 min; total time of analysis 59 min, equilibration evaluated for the first time by using noninvasive in vivo time of 10 min, and a flow rate of 0.4 mL/min. The column imaging technique. was a Poroshell 120 EC18 (150 × 4.6 mm i.d., 2.7 m) with ∘ a precolumn of the same phase maintained at 27 C; the 2. Methods and Materials eluents were H2O (pH 3.2 by HCOOH) and CH3CN, both of HPLC grade. The HPLC/ESI/MS analysis was carried out 2.1. Plant Material. Mature grapes were collected from dif- using a liquid chromatographic HP 1100 L equipped with an ferent cultivars of Vitis vinifera L.: Michele Palieri (M. Electrospray (ESI) HP 1100 MDS mass detector with an API interface. The operative conditions of the mass spectrometer Palieri), Italia, Red Globe, Negroamaro, Pinot, Abbuoto, −1 were nitrogen flux 10 L min , nebulizer pressure 30 psi, and Verdicchio. The cultivars M. Palieri, Italia, Red Globe, ∘ ∘ and Negroamaro were grown in the experimental farm of gas temperature 350 C, quadruple temperature 30 C, and CRA-UTV in Turi (BA), during 2010 and 2011 with the capillary voltage 3000–4000 V.The experiments were carried “tendone” system, a typical cultivation method in the Puglia out in negative and positive ionization modes, applying frag- region (South Italy) whose climate is characterized by scarce mentors between 60 and 220 V.The following standards were − rainfalls [12]. The vines were treated with reduced irrigation used for the identification: (+) catechin, ( )epicatechin,ECg, 3 3 volume per hectare (1200 m )(V1)or(2000m)(V2)and procyanidin B1, and procyanidin B2, all of high purity grade −1 with reduced nitrogen fertilization (120 kg ha )(N1)orwith and purchased from Extrasynthese (France). The quantitative −1 180 kg ha (N2) that is the quantity generally used in the analysis of both phenol oligomers and polymers was carried out at 280 nm using only the procyanidin B2 as external growing area. Fertilization was carried out at budding (mid- March) and during the growth of the green grapes (first standard in a concentration range 0.1–5.7 gandafive-point 2 ten days of July). The cultivars Verdicchio, Abbuoto, and calibration curve with of 0.999. Pinot were grown in the experimental field of Lazio region (Center of Italy), during vintages 2006, 2008, and 2011 in 2.4.