Evaluation of Anti-Candida Activity of Vitis vinifera L. Seed Extracts Obtained from Wine and Table Cultivars

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 on 43 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 be used in mucosal infection as vaginal candidiasis.


Introduction
Candida species are major human opportunistic fungal pathogens that cause both mucosal and deep tissue infections. The frequency of mucosal and cutaneous fungal infections has dramatically increased worldwide. Infection caused by Candida spp. affects 70-75% of women at least once during their life. Recurrent vulvovaginal candidiasis occurs in 5% of women with Candida vaginitis [1,2]. Most of these infections are caused by Candida albicans (C. albicans) and among nonalbicans Candida spp., C. glabrata, C. tropicalis, and C. krusei.
Azoles are the most common antifungal agents available to treat topical Candida infections. However, these antifungal drugs have several defects related to clinical usage, such as low efficacy and side effects. Therefore, there is an urgent need of new antifungal agents [3]. Natural anti-infective agents represent a promising approach for the treatment of Candida infections [4]. Phytomedicine, which has historically been an important aspect of traditional medicine in nonindustrialized countries, is now becoming an integral part of healthcare in industrialized countries. Plants are the source of thousands of new phytochemicals, and different strategies can be applied to 2 BioMed Research International improve the yields of bioactive metabolites in the plant and to obtain chemically standardized extracts [5,6]. Vitis vinifera L. is the most important fruit species in the world, cultivated especially in Mediterranean area. As reported by the wide literature [7], grapes are rich source of polyphenols, important secondary metabolites produced by higher plants, which play multiple essential roles in plant physiology and showed healthy properties in human organism, mainly as antioxidant, antiallergic, anti-inflammatory, anticancer, antihypertensive, renoprotective, and antimicrobial agents [8,9]. GSEs are recognized as a complex mixture of monomeric, oligomeric, and polymeric flavan-3-ols. The principal monomers identified are (+)-catechin, (−)-epicatechin, (−)-epicatechin gallate (ECg), (−)-epigallocatechin (EGC), and (−)-epigallocatechin gallate (EGCg). Several fungi, including C. albicans, are sensitive to EGCg, the main component of green tea extracts [10]. The content of flavan-3-ols in seed grapes is influenced by several factors mainly cultivar, irrigation, nitrogen fertilization, delayed harvest, and storage conditions [11].
Moreover, the application of an extraction process suitable to efficiently recover the target metabolites and an appropriate analytical method for an accurate qualitative and quantitative determination of extract components are required.
In this work, for the first time, anti-Candida activity and chemical analysis of GSEs obtained from wine and table cultivars of Vitis vinifera L. grown in different agronomic conditions have been evaluated and compared with respect to their phenolic content. The HPLC method with a Poroshell column has allowed to quantify not only the flavan-3-ols oligomers but also the polymeric forms (polymerization degree >4) difficult to be detected with conventional reverse phase columns. Moreover, the effect of GSE treatment on an experimental murine model of vaginal candidiasis was evaluated for the first time by using noninvasive in vivo imaging technique.

Methods and Materials
2.1. Plant Material. Mature grapes were collected from different cultivars of Vitis vinifera L.: Michele Palieri (M. Palieri), Italia, Red Globe, Negroamaro, Pinot, Abbuoto, and Verdicchio. The cultivars M. Palieri, Italia, Red Globe, and Negroamaro were grown in the experimental farm of CRA-UTV in Turi (BA), during 2010 and 2011 with the "tendone" system, a typical cultivation method in the Puglia region (South Italy) whose climate is characterized by scarce rainfalls [12]. The vines were treated with reduced irrigation volume per hectare (1200 m 3 ) (V1) or (2000 m 3 ) (V2) and with reduced nitrogen fertilization (120 kg ha −1 ) (N1) or with 180 kg ha −1 (N2) that is the quantity generally used in the growing area. Fertilization was carried out at budding (mid-March) and during the growth of the green grapes (first ten days of July). The cultivars Verdicchio, Abbuoto, and Pinot were grown in the experimental field of Lazio region (Center of Italy), during vintages 2006, 2008, and 2011 in normal Mediterranean conditions. The average amount of rainfall accumulated between April and September in 2006, in 2008, and in 2011 has been 223 mm, 423 mm, and 245 mm, respectively. The cultivars had training system to Cordon Spur, with plant density of 2.60 × 1 m. In the examined years the same cultural practices were applied in the vineyard. All the grapes were harvested at technological maturation and frozen at −20 ∘ C. The seeds have been isolated immediately before use and subjected to extraction process.

Sample Preparation.
The seeds were separated from the flesh and the skin, weighed, and put in liquid nitrogen in a porcelain mortar and ground to obtain a fine powder. They were extracted three times (24 hrs for each extraction) by the mixture EtOH/H 2 O (7 : 3 v/v) acidified with formic acid to pH 3; the ratio matrix/solvent was 1 g fresh weight/10 mL. After the removal of the solid residue, the extracts were dried (t ≤ 30 ∘ C), weighed, and redissolved in a suitable volume of the same extraction solution to obtain enriched extracts. The samples were centrifuged (12.000 rpm for 5 min) to obtain a limpid solution for the HPLC/DAD/MS analyses. Only the seeds of Abbuoto and Verdicchio cultivars (vintages 2006 and 2008) were treated with a different method using a buffer at pH 3.2 as extractive solution. This method was applied with the aim to simulate the wine-making process. The seeds were manually separated from the berries and extracted with 125 mL of the buffer solution for 144 hours at 30 ∘ C. The buffer composition consisted of tartaric acid 5 g; NaOH 1 N 22 mL; Na 2 S 2 O 5 2 g; and EtOH 95% 120 mL. O for 4 min and a plateau for 5 min; total time of analysis 59 min, equilibration time of 10 min, and a flow rate of 0.4 mL/min. The column 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 eluents were H 2 O (pH 3.2 by HCOOH) and CH 3 CN, both of HPLC grade. The HPLC/ESI/MS analysis was carried out using a liquid chromatographic HP 1100 L equipped with an Electrospray (ESI) HP 1100 MDS mass detector with an API interface. The operative conditions of the mass spectrometer were nitrogen flux 10 L min −1 , nebulizer pressure 30 psi, gas temperature 350 ∘ C, quadruple temperature 30 ∘ C, and capillary voltage 3000-4000 V. The experiments were carried out in negative and positive ionization modes, applying fragmentors between 60 and 220 V. The following standards were used for the identification: (+) catechin, (−) epicatechin, ECg, procyanidin B1, and procyanidin B2, all of high purity grade and purchased from Extrasynthese (France). The quantitative analysis of both phenol oligomers and polymers was carried out at 280 nm using only the procyanidin B2 as external standard in a concentration range 0.1-5.7 g and a five-point calibration curve with 2 of 0.999.  [13]. The gLUC59 luciferase reporter has previously been described [13]. C. albicans gLUC59 and the control strain were cultured in YPD as previously described [14].

Antifungal Susceptibility
Testing. The broth microdilution method to evaluate the susceptibility in vitro on strains of Candida spp. was performed according to standardized method for yeast [15,16]. Briefly, the extracts were dissolved previously in dimethyl sulfoxide at concentrations 100 times higher than the highest test concentration [16]. The final concentration ranged from 0.25 to 512 for total dry seed extracts and from 0.125 to 128 mg/L for Fluconazole (FLC). Microdilution trays, containing 100 L of serial twofold dilutions of seed extracts or FLC in RPMI 1640 medium (Sigma-Aldrich, St. Louis, MI, USA), were inoculated with an organism suspension of 1.0 × 10 3 -1.5 × 10 3 cells/mL. The panels were incubated at 35 ∘ C and the growth observed at 48 h. The minimal inhibitory concentration (MIC) was the lowest concentration that caused a prominent decrease (≥50%) in visible growth. The MIC 90 was defined as the lowest drug concentration that caused ≥90% growth inhibition compared with the drug-free control. Medians, geometric means (GMs), and ranges were calculated.
2.6. In Vitro Induction of Resistance. C. albicans ATCC 10231 was cultured as previously described [17]. In particular, serial subcultures of C. albicans ATCC 10231 were performed in fresh medium every 48 hours, containing rising concentrations of M. Palieri V1N1 2010 GSE (starting from 4 mg/L up to 128 mg/L). At the same time the same procedure was carried out for the control without addition of extract. The MIC was evaluated after 48 h of incubation (extracts concentration range 0.5-512 mg/L) according to the CLSI protocol [15].

Mice. Female CD1 mice obtained from Harlan Nossan
Laboratories (Milan, Italy) were used at 4 to 6 weeks of age. Mice were allowed to rest for 1 week before the experiment; by that time the animals were roughly 5 to 7 weeks old. Mice were used under specific pathogen-free conditions that included testing sentinels for unwanted infections; according to the Federation of European Laboratory Animal Science Association standards, no infections were detected.

Infection and Treatment.
Mice infection was performed as previously described with minor adaptations [18]. Mice were maintained under pseudoestrus condition by subcutaneous injection of 0.2 mg of estradiol valerate in 100 L of sesame oil (Sigma-Aldrich) 5 days prior to infection and weekly until the completion of the study. Mice anaesthetized with 2.5-3.5 (v/v) isofluorane gas were infected with 10 L of 2 × 10 9 cell/mL of C. albicans gLUC59 or the control strain. Cell suspensions were administered from a mechanical pipette into the vaginal lumen close to the cervix. To favor vaginal contact and adsorption of fungal cells, mice were held head down for 1 min following inoculation. Mice were then allowed to recover for 24-48 h, during which the Candida infection was established. The intravaginal treatment with FLC (200 mg/L, 10 L/mouse) or with M. Palieri V1N1 2010 GSE (50 mg/mL, 10 L/mouse) has begun 2 h before the challenge and then it was repeated every two days until day +8. isofluorane. Total photon emission from vaginal areas within the images (region of interest (ROI)) of each mouse was quantified with Living ImageR software package [19].

Statistical
Analysis. In order to analyze the data among phenolic constituents and MIC values of dried GSEs obtained from selected cultivars of Vitis vinifera L., Pearson's correlation coefficient (r) was determined. A value of <0.01 was considered significant. Differences between FLC and M. Palieri V1N1 2010 GSE treated and saline treated mice were evaluated by Mann-Whitney U-test. A value of < 0.05 was considered significant.     Table 5).

Chemical Composition of the Extracts.
An example of the GSEs HPLC profiles in terms of monomers, oligomers, and polymers is shown in Figure 1. Optimizing the chromatographic method also with the help of an RP 18 Poroshell column several monomer and oligomer compounds have been separated and quantified in all the fifteen extracts ( Table 5). The identification of the phenols listed in Table 5 has been done by mean of their UV and mass spectra, by the use of pure standards ((+) catechin; (−) epicatechin; procyanidin B1 and B2; ECg) and according to the literature [20][21][22]. Moreover, two groups of polymeric procyanidins (Pol 1 and Pol 2) with a polymerization degree ≥4 have been separated and determined by their mass spectra in negativeionization mode (Figure 1 and Table 5). The chemical composition of the different extracts correlated to their antifungal activity against C. albicans reference strains is summarized in Table 5. The identified components in the GSEs typically belong to the flavan-3-ols class; (+) catechin and (−) epicatechin are the main monomers; within the polymeric forms 1 + 2 (from 40 to 96% of the total flavan-3-ols) are included some acylated forms with gallic acid residues. The extracts 1-4 and 15 (Table 5) showed the lowest content of flavan-3-ols and in particular the lowest content of polymeric forms compared with all the other samples (Table 5).  Antifungal activity of M. Palieri V1N1 2010 GSE against vaginal candidiasis was monitored in an experimental murine model of vaginal candidiasis that allowed the visualization of the temporal and spatial progression of infection. In particular the M. Palieri V1N1 2010 GSE was administered intravaginally (50 g/L, 10 L/mouse) 2 h before the challenge and then it was repeated every two days until day +8. The results reported in Figure 2 showed a significant inhibition of C. albicans load observed 5 days after challenge. The effect was comparable to that observed with FLC. The infection was completely cleared 8 days after infection (Figure 2).

Discussion
In this work, for the first time, the in vitro anti-Candida activity of GSE from wine and table cultivars of Vitis vinifera L., grown in different agronomic conditions, collected over several years has been evaluated. The results showed that GSEs obtained from cultivars grown in Puglia under hydric stress possess potent antifungal activity in vitro, in some cases similar to that of FLC itself. All GSEs have been chemically characterized. A significant negative correlation coefficient of total flavan-3-ols contained in the different extracts and MIC values has been demonstrated ( = −0.648, = 0.00896). Moreover, we demonstrated for the first time that the antifungal activity (MIC) of GSEs is attributable mostly to the polymeric flavan-3-ols (with a polymerization degree ≥4), with a significative negative correlation coefficient ( = −0.6974, = 0.0038) ( Table 5). Differently, the content of gallate monomers and oligomers did not seem to be correlated to antifungal activity ( = −0.4334, = 0.1065). It is important to emphasize that the typical catechin of green tea, EGCg, known to be responsible of growth-inhibitory effect on clinical isolates of Candida spp. [10], is absent in our samples.
We demonstrated that Verdicchio and Abbuoto, not subjected to controlled agronomic conditions of water, collected in 2006 (samples 1-2), had a higher content of polymers 1 + 2 (with the polymer 2 from 3-to 10-fold higher) than the same cultivars collected in 2008 (Table 5). These differences could be partially attributable to the rainfall during 2008, twofold higher than that in 2006.
Hydric stress implies a bigger effort to absorb water from the soil and, as a consequence, a lesser vegetative growth and an increase of bioactive molecules production [23]. In particular, Cavaliere et al. demonstrated that the content of flavan-3-ols in grape seeds is influenced by several agronomic conditions mainly irrigation and nitrogen fertilization [11,24].
In the present work, the dried GSEs obtained from the cultivars M. Palieri, Italia, Red Globe, and Negroamaro, cultured under hydric stress (V1 and V2) (samples 5-14 Table 5), showed the highest content of polymers 1 + 2 >250 mg/g with the polymer 2 >46 mg/g and the best antifungal activity with  the GM MIC values between 5.7 and 20.2 mg/L against C. albicans reference strains (Table 5) as reported in the patent PCT/IT2011/000400 [25]. GSE, with 573 mg/g of polymeric flavan-3-ols, obtained from M. Palieri V1N1 2010, has been tested in an experimental murine model of vaginal candidiasis, by using noninvasive in vivo imaging technique. This GSE demonstrated to accelerate the clearance of fungus during vaginal candidiasis, with a significant inhibition of C. albicans load 5 days after challenge, evaluated by photon emission.

Conclusion
In conclusion, we demonstrated that GSEs obtained from Vitis vinifera plants, grown under hydric stress, had a high and reproducible content of polymeric flavan-3-ols, with a polymerization degree ≥4, and high antifungal activity. Further studies are in progress to characterize these polymeric fractions.
Moreover, we demonstrated, for the first time, anti-Candida activity of GSE in an experimental murine model of vaginal candidiasis.
These findings, together with lack of toxicity and easy way of preparation of the extracts, suggest that GSEs with high content of polymeric flavan-3-ols could be used in Candida infections.

Ethical Approval
All animal experiments were performed in agreement with the EU Directive 2010/63, the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes, and the National Law 116/92. The protocol was approved by Perugia University Ethics Committee for Animal Care and Use (Permit no. 149/2009-B). All the animals were housed in the animal facility of the University of Perugia (Authorization no. 34/2003A) in microisolator boxes with autoclaved diets and wood chips. Mice were acclimatized for a week before starting the experiments. All efforts were made to minimize suffering during experiments.

Conflict of Interests
All the authors declare that they had no personal relationship with other people and organisations that could inappropriately influence our work. G. Pasqua, G. Simonetti, F. D. D' Auria, A. R. Santamaria, D. Antonacci, and N. Mulinacci are named inventors on a patent pending based on italian priority IT RM20100636 and related to "Extracts obtained from Vitis vinifera seeds and/or pomace and/or green grapes and/or stalks and uses thereof as antifungal agents" PCT/IT2011/000400, which is owned by Sapienza University (70%), CRA (20%), and University of Florence (10%). No commercial agreements are presently in place.