Antioxidant Effect of the Castanea sativa Mill. Leaf Extract on Oxidative Stress Induced upon Human Spermatozoa

This study was aimed at evaluating in vitro the effects of a 75% v/v ethanolic extract of leaves of Castanea sativa Mill. (var. Bastarda Rossa, Mount Amiata, Tuscany, Italy) on ejaculated human sperm. Total polyphenols and total flavonoids contained in the extract were determined by a colorimetric assay and HPLC-DAD. The DPPH test and electrochemistry were utilized to study the antioxidant activity of the extract. Swim-up-selected sperm from 8 healthy men were treated with the C. sativa leaf extract at different dilutions (1 : 100, 1 : 200, and 1 : 500), and sperm motility was assessed following the WHO guidelines. Swim-up-selected spermatozoa were incubated with 100 μM H2O2 to induce lipid peroxidation (LPO) and with H2O2 and the leaf extract (1 : 100, 1 : 200, and 1 : 500) to test the antioxidant activity of the extract. The levels of LPO were determined by measuring malondialdehyde (MDA) concentrations. The treated samples were also analyzed by transmission electron microscopy (TEM) for ultrastructural evaluation. The chemical analysis showed that one-third ca. of the polyphenols in the C. sativa extract were made up of flavonoids, with hyperoside present in high concentration. A good antioxidant activity was demonstrated by both the DPPH test and electrochemical analysis. The C. sativa leaf extract did not decrease sperm motility at all tested dilutions. Treatment with H2O2 alone caused a significant increment in MDA levels (P = 0.006993), while the treatment with H2O2 plus C. sativa extract diluted to 1 : 100 and 1 : 200 significantly reduced MDA levels (P = 0.01476 and P = 0.01571, respectively), with respect to H2O2 alone. TEM analysis confirmed the protective effect of the extract on damage induced by LPO, in particular that occurring at the plasma membrane level. The C. sativa leaf extract could be used in human and farm animal protocols for gamete handling, such as techniques of assisted reproduction and cryopreservation of semen, all conditions in which oxidative stress is exacerbated.


Introduction
Castanea sativa Mill. (C. sativa) is a very long-lived tree, belonging to the Fagaceae family present in South Europe and Asia. The fruit chestnut is a healthy food and has valuable nutritional qualities. It is noteworthy that when harvesting chestnuts, a great deal of waste products is obtained, such as leaves and inner and outer shells. Therefore, it could be important to try to use also the waste material to find substances that can be utilized in industrial, cosmetic, and nutra-ceutical fields and other areas, encouraging recycling and a sustainable economy [1].
Extracts of this plant have displayed a clear biological activity; in particular, they have shown cardioprotective [2], antihelminthic, antibacterial, and antiviral effects [3] and neuroprotective activity [4]. Observations recently made by Budriesi et al. [5] in high-fat diet rats suggested that the chestnut extract has the potential to be used in medicine as a dietary supplement in the treatment of obesity complications. For a long time, chestnut leaf infusions have been used to treat rheumatism, cough, and diarrhea [6]. They also exert calming and sedative action and are an excellent remedy in the treatment of cold and cases of respiratory problems.
Recent studies highlighted the presence of high concentrations of phenolic compounds endowed with antioxidant activity in the chestnut waste material extract [1]. Leaves are rich in tannins (6%-8% of leaf dry weight) including ellagic acid, as well as flavonoids (0.1-0.3%) such as rutin, hesperidin, quercetin, apigenin, morin, galangin, and kaempferol [3,7]. Cinnamic derivatives, such as chlorogenic acid, are also present [7]. It is known that these compounds are involved in the defense against the oxidative damage and are able to protect the main targets of reactive oxygen species (ROS), which are DNA, proteins, and lipids [8]. The scavenging activity of these compounds is due to the presence of hydroxyl groups [9]. Therefore, C. sativa leaf extracts are used in different applications. For example, they are able to protect from oxidative damage the DNA of pancreatic β-cells treated in vitro with the diabetogenic agent streptozotocin [10]. Quave et al. [11] reported that C. sativa leaf extracts rich in pentacyclic triterpenes, such as ursene and oleanane derivatives, blocked the Staphylococcus aureus pathogenic activity and virulence. In addition, leaf phenolic extracts of the Italian product Marrone di Roccadaspide (Campania region, Italy) showed a protective effect against UVB-induced damage on human keratinocytes [12].
It is known that oxidative stress results from alterations in the complicated balance between reactive oxygen species (ROS) generation and elimination. The presence of physiological levels of ROS supports some main functions of sperm, such as motility, capacitation, acrosome reaction, zona pellucida binding, and oocyte fusion [13]. However, an uncontrolled ROS production and/or the defects in the balance between ROS concentration and the antioxidant scavenging system affect the normal sperm function damaging sperm membranes, DNA, and proteins [14]. In particular, sperm membranes are rich in polyunsaturated fatty acids (PUFAs), and they are vulnerable to ROS that causes lipid peroxidation (LPO) [15].
Sperm and seminal plasma are endowed with enzymatic and nonenzymatic antioxidant compounds able to scavenge ROS [16]. However, semen laboratory processing such as centrifugation, cryopreservation, and, in general, semen handling leads to an increase in ROS production [17,18]. A strategy to resolve this problem encompasses the supplementation of used media with antioxidants. With this purpose, many in vitro studies reported the scavenging ability of antioxidant compounds against oxidative stress induced to human sperm [19][20][21][22][23].
The aim of this study was to characterize the antioxidant potential of the extract of Castanea sativa Mill. (Bastarda Rossa) leaves and to evaluate its effect on oxidative stress induced upon human spermatozoa. The chemical analysis, performed by high-performance liquid chromatographydiode array detection (HPLC-DAD), enables the evaluation of the flavonoid and polyphenol content. The antiradical capacity was tested by the DPPH assay and electrochemical measurements. The effects of the C. sativa leaf extract were then assayed upon ejaculated human sperm in in vitro experiments. The possible toxicity of this extract was assessed by determining its effect at different dilutions on the sperm motility. The antioxidant ability of the extract against LPO [induced by hydrogen peroxide (H 2 O 2 )] was evaluated by measuring the levels of malondialdehyde (MDA) in human swim-up-selected sperm treated with H 2 O 2 alone and H 2 O 2 plus C. sativa extract. The morphological alterations induced by H 2 O 2 upon spermatozoa and the protective effect of the extract were analyzed also by transmission electron microscopy (TEM).

Materials and Methods
2.1. C. sativa Extract Preparation. Castanea sativa Mill. var. Bastarda Rossa leaves were harvested in Arcidosso (Mount Amiata, Tuscany, Italy) in August, during their full development stage. Collection was performed in the morning, and leaves were manually chosen when they are intact and healthy and have no signs of microbial infection. Herbal material was dried to constant weight at 35°C.
The extraction procedure was accomplished using the automatic extractor Naviglio Estrattore® (Atlas Filtri, Padova, Italy) and validated and was very effective in obtaining an optimal yield of polyphenols from herbal drugs [24]. Briefly, dried C. sativa leaves were crushed and transferred into a 50 μm extraction bag up to complete filling. The bag was placed in the extraction chamber that was filled with solvent of extraction (ethanol 75% v/v). Naviglio Estrattore® parameters were set as follows: static phase: 8 min/cycle, dynamic phase: 18 laps/cycle, and total cycles of extraction: 100. At the end of the extraction procedure, the extract was adjusted to a final drug : extract ratio 1 : 10.

Chemical Analysis.
Total polyphenol and flavonoid content of the ethanolic extract (CEE) was examined using spectrophotometric methods reported in Biagi et al. [24]. In detail, total polyphenols were determined by the colorimetric method of Folin-Ciocalteu: 0.1 ml of each extract was added to 2.9 ml of distilled water and 0.5 ml of Folin-Ciocalteu reactive (1 : 10 v/v) to distilled water.
After 30 s of shaking, 1 ml of Na 2 CO 3 15% w/w was added to distilled water. After incubation at room temperature for 120 min, the absorbance at 700 nm was read using a SAFAS UV-MC2 instrument (Monte Carlo, Monaco). The polyphenol quantification was calculated by means of interpolation of the calibration curve constructed using gallic acid (Sigma-Aldrich, Milan, Italy).
Total flavonoid content of extracts was determined reading the absorbance at 353 nm of the extract diluted to 100-fold, according to Sosa et al. [25], and constructing a calibration curve using hyperoside as the standard (Sigma-Aldrich, Milan, Italy). All reagents were purchased from Sigma-Aldrich (Milan, Italy). A HPLC-DAD analysis was carried out to investigate the polyphenolic fraction of CEE and to identify the main chemical constituents. A Shimadzu Prominence LC 2030 3D instrument equipped with a Bondapak® C18 column (10 mm, 125 Å, 3:9 mm × 300 mm column) (Waters Corporation, USA) was used.
Water+0.1% v/v formic acid (A) and acetonitrile+0.1% v/v formic acid (B) were used as mobile phases. The following program was applied: B from 10% at 0 min to 25% at 20 min and then B 50% at 35 min; the flow was set at 0.8 ml/min. Chromatograms were recorded at 280 nm and 340 nm.
Analyses were performed using 10 μl of CEE; hyperoside and quercetin (Sigma-Aldrich, Milan, Italy) were used as external standards. Calibration curves were established using reference standards ranging from 0.008 mg/ml to 0.5 mg/ml. The correlation coefficient (R 2 ) of each curve was >0.99.
2.3. Antiradical Capacity: DPPH Assay. The antiradical capacity of CEE was tested by means of the validated DPPH (2,2-diphenyl-1-picrylhydrazyl) test. All reagents were purchased from Sigma-Aldrich, Milan, Italy. The DPPH solution was prepared in methanol at a concentration of 1 × 10 −4 M. CEE was tested in seven 1 : 2 serial dilutions (ethanol 75% v/v), ascorbic acid, used as the reference, was dissolved in pure water, and hyperoside and quercetin were dissolved in ethanol 75% v/v and diluted in order to obtain a range of concentrations from 0.031 to 2 mg/ml. All the samples were mixed with the DPPH solution (1 : 19), transferred into 1 cm path length cuvettes, and incubated for 30 min at room temperature in the dark. Water or ethanol 75% v/v in DPPH (1 : 19) was used as the positive control. The inhibition of DPPH was calculated according to the following formula: where Abs c is the absorbance of the positive control and Abs e is the absorbance of the tested samples. IC 50 was calculated by constructing the curve of inhibition values for each tested concentration (in the linear range 10-75%) [22].

Electrochemical Measurements.
Electrochemical experiments (cyclic voltammetry and differential pulse voltammetry (DPV)) were carried out to obtain information about redox properties of CEE as well as also of its main flavonoids such as hyperoside and quercetin. The electrochemical analyzer BAS100A (Bioanalytical Systems Inc., West Lafayette, USA) connected to a conventional three-electrode cell consisting of a glassy carbon working electrode, a platinum wire as an auxiliary electrode, and an Ag/AgCl reference electrode was used. The reported potentials were referred at a scan rate of 20 mV/s. Measures were acquired at room temperature under an ultrapure nitrogen inert atmosphere to prevent both the reactivity of the examined samples with the atmospheric oxygen and the dissolved oxygen reduction process. This analysis was carried out as follows:

Semen Samples.
Semen samples from 8 healthy men (aged 24-30 years) were collected by masturbation after 3-5 days of sexual abstinence. They were analyzed after liquefaction for 30 min at 37°C. Volume, pH, concentration, and motility were evaluated according to the World Health Organization guidelines [26]. All men provided an informed written consent before the inclusion in this study. The informed consent described the aims of the research and was approved by the Ethics Committee of Azienda Ospedaliera Universitaria Senese (CEAOUS).
2.6. Swim-Up to Select Motile Sperm. A swim-up technique was used to obtain the motile sperm fraction from 8 different ejaculates: 0.5 ml of each semen sample was placed in a sterile conical centrifuge tube and gently layered with 0.5 ml of Biggers-Whitten-Whittingham (BWW) medium [26]. The tubes, inclined at a 45°angle, were incubated for 45 min at 37°C and 5% CO 2 . Then, 0.5 ml of the uppermost medium that contains motile sperm fraction was collected and used for the experiments.

Determination of Sperm Motility after C. sativa Leaf
Extract Treatment. Different dilutions of C. sativa leaf extracts in BWW medium (1 : 100, 1 : 200, and 1 : 500) were added to swim-up-selected sperm. Mixtures were incubated at 37°C and 5% CO 2 for 1 h. Sperm motility was evaluated using a Burker counting chamber. Spermatozoa were categorized in different grades of motility (sperm with progressive and nonprogressive motility and immotile sperm) [26]. Aliquots of the selected sperm treated in the same conditions but without the C. sativa leaf extract were used as controls. All experiments were carried out eight times, and results were reported as mean values ± standard deviation (SD) and median.

Results
3.1. Chemical Analysis of C. sativa leaf extract. The Folin-Ciocalteu colorimetric assay and direct spectrophotometry enabled the quantification of the total polyphenols and total flavonoids in CEE, respectively. Table 1 shows the quantifications of total polyphenols expressed as gallic acid equivalents (mg/l of extract) and total flavonoids expressed as hyperoside (mg/l of extract). Results showed that about one-third of the polyphenols in the extract are flavonoid type; it was plausible to refer the remaining polyphenolic portion to phenolic acids, hydroxycinnamic derivatives, and tannins.
HPLC From the comparison with the standards and the literature, it was possible to assign the peak at 18.25 min to the hyperoside (Figure 1), while the peak at 18.13 min is with high probability an isomer of the hyperoside, plausibly isoquercitrin. Quercetin is also present in the extract, but in low concentration (RT = 25:09 min, 6.30 mg/l). The main nonflavonoid constituent can be assigned to the catechin tannin class, but it was not identifiable not being referable to the used reference standards. In CEE, the concentration of hyperoside was 75.28 mg/l.

Antiradical
Capacity: DPPH Assay. The DPPH assay was carried out on CEE to measure its antiradical capacity. Free radical inhibition values were linearly correlated with CEE concentration in the concentration range 0.05%-0.2%, while at higher concentrations, the typical maximal inhibition plateau was obtained.
From the construction of the correlation line between concentration and activity in the linear zone, it was possible to calculate the IC 50 (inhibitor concentration that reduces the activity by 50%) of CEE, which was found to be 0.081% (0.72 mg/ml). Results were validated using ascorbic acid as the reference IC 50 < 10 μg/ml [28]. The CEE phytocomplex exerted a synergistic effect compared to the main single compounds. Indeed, repeating the DPPH test on hyperoside and quercetin, we found that the best IC 50 value was exerted by quercetin 0.01 mg/ml, while the IC 50 value of hyperoside was 0.06 mg/ml; these values were higher than those which one would expect considering the presence of these compounds at the IC 50 value of CEE (when CEE diluted at 0.081% contains 0.06 mg/l of hyperoside and 0.005 mg/l of quercetin).

Electrochemical Measurements.
Cyclic voltammograms recorded in the solution containing quercetin showed a quasireversible oxidation process with an anodic peak at +0.33 V. The same profile was recorded in the solution containing hyperoside, but the anodic peak potential was reached at +0.44 V (data not shown in figures). More complicated redox behaviour was recorded in the extract solution. Indeed, the cyclic voltammogram of the CEE solution ( Figure 2, curve b arrow) showed a main irreversible oxidation process at +0.55 V; however, the differential pulse voltammetry, analysis more sensitive than cyclic voltammetry, highlighted that CEE undergoes two other cathodic processes with peak potentials around +0.15 V and +0.40 V (Figure 2, curve a asterisks). It is interesting to note that both peak values are lower than that measured in hyperoside solution (+0.44 V) supporting the highest antioxidant activity recorded for CEE.

Discussion
In this research, we characterized a 75% v/v ethanolic leaf extract of a typical Tuscan chestnut variety: "Bastarda Rossa" from Mount Amiata. C. sativa leaves are the least exploited by-product of this species, despite the fact that they are rich in phenolic compounds, which are endowed with antibacterial and DNA-protective activities and are efficient in the prevention and treatment of oxidative stress-mediated diseases such as photoaging [29]. A 70% v/v ethanolic extract of C. sativa leaves from Mirandela (Portugal) was investigated and resulted to be very effective in in vitro scavenging and antioxidant tests. In a cutaneous irritation test, performed in 20 healthy people, it resulted to be well tolerated and free from side effects [30]. Barreira et al. [31], in a comparative study, reported that antioxidant potential of chestnut leaves was higher than that of almond leaf extracts.
Chemical analysis conducted in this work showed that CEE contains a concentration of total polyphenols (3475.23 mg/l) higher than that of other well-studied antioxidant natural products such as red wine [32] and green and black teas from Camellia sinensis Kuntze leaves [33]. Interestingly, a large part of these polyphenols is represented by monomeric flavonoids; in particular, hyperoside and quercetin glycosides were the most abundant constituents. Recently, great attention has been focused on hyperoside for its effective antioxidant capacity in an innovative model of oxidative stress using the yeast Saccharomyces cerevisiae [34]. Hyperoside also showed cytoprotective action against H 2 O 2 -induced cell damage in Chinese hamster lung fibroblast cells, exerting intracellular scavenging activity [35]. In addition, quercetin and its main glycosides, such as rutin and isoquercitrin, are well-known antioxidant molecules [36]. By monitoring the qualitative profile of the recorded chromatogram and according to Cerulli et al. [12] who studied the methanol extract of the leaves of C. sativa var. "Marrone di Roccadaspide," it is plausible to refer that phenolic acids, hydroxycinnamic derivatives, and tannins are also present in the CEE. To determine the antioxidant capacity of the chestnut leaf extract, we employed either the classic and validated DPPH test or the cyclic voltammetry, an interesting, but least exploited, technique. CEE exerted a good antiradicalic capacity in the DPPH test, showing to be effective even diluted to over 1000-fold. The IC 50 value of the extract is comparable with that of a polyphenol-enriched red wine [37]. In spite of its simplicity, the qualitative conventional electroanalysis has a prominent role in the assessment of antioxidants in food and biological samples and in the evaluation of their antioxidant activity in vitro. The relationship between the sample voltammogram and its in vitro antioxidant activity was defined by its oxidation potential: peaks with low values of oxidation potential were associated with high antioxidant activity while peaks with the high values were associated with low antioxidant capacities. The high sensitivity of DPV enables pointing out processes with low peak current, which is difficult to highlight with cyclic voltammetry. In our extract, these peaks were present at peak potential values of +0.15 to +0.40 V and +0.55 V, with the last one evident also in the cyclic voltammogram. All these potential peaks are associated with a good antioxidant activity according to the electrochemical behaviour typical of flavonoids [38].
In particular, the peaks located at more cathodic potential values than quercetin and hyperoside indicate that the chemical composition of the extract includes minor components which are responsible for the antioxidant activity of CEE.
These findings related to the redox characteristics of the chestnut extract implemented those obtained in the evaluation of antiradicalic capacity with DPPH and highlighted the antioxidant characteristics of the extract phytocomplex.
The experimental protocol performed in this work confirmed that the chestnut leaf extract of Bastarda Rossa is worthy to be investigated for its interesting biological characteristics. For this reason, we decided to explore the ability of the C. sativa leaf extract to prevent LPO induced in vitro by H 2 O 2 on swim-up-selected human sperm. Our final purpose was to find out natural substances that could be used as antioxidant supplementations in fertilization culture media.
It is known that semen processing for assisted reproduction technologies and cryopreservation requires protocols that include centrifugation and incubation of spermatozoa in culture media and that these procedures could exacerbate oxidative stress and induce damage to sperm cells [39] due to ROS generation. Spermatozoa, particularly human ones, are vulnerable to ROS attack. ROS may cause LPO of sperm membranes given that they are particularly rich in unsaturated fatty acids. The LPO process leads to a loss of membrane integrity, an increase in its permeability, inactivation of cellular enzymes, DNA damage, and cell apoptosis. This problem is not only related to semen handling, since it is estimated that semen samples from 25% ca. of infertile men present elevated levels of ROS and, often, decreased antioxidant capacity [40]. The oral supplementation of antioxidant substances may improve semen parameters and oxidant/antioxidant status in infertile males [41]. Therefore, it seems reasonable to support the treatment of male infertility with substances able to neutralize ROS. In this particular field, the study of natural compounds endowed with antioxidant properties is appealing, either from the point of view of male infertility treatment or for the development of new strategies for the supplementation in vitro of media used for semen handling.
We have previously demonstrated the scavenging activity of some natural compounds, such as resveratrol, quercetin, rutin, naringenin, epicatechin, and Propolfenol®, a phytocomplex rich in European propolis and catechins, against LPO induced in vitro to human spermatozoa [20][21][22]42]  effect of hydroxytyrosol, the main phenolic compound of virgin oil, during sperm centrifugation. In addition, in a mouse model, it was found that the supplementation of media with antioxidants during all stages of the in vitro fertilization procedures and cultures could play a beneficial effect on assisted reproduction and may preserve embryo viability [43].
The results of this study demonstrated that the C. sativa leaf extract used at dilutions of 1 : 100, 1 : 200, and 1 : 500 did not show any toxicity on sperm motility. These results are different from those obtained by some of our group using purified polyphenols [20,21], which, at high concentration, decreased sperm motility and viability and damaged DNA, as we observed using liposomes loaded with 100 μM quercetin [42].
It seems that a combination of natural antioxidant compounds is more tolerated than single purified compounds and that the phytocomplex exerts a synergistic action, at least in this kind of in vitro experiments, as it was observed also when we used Propolfenol® in similar protocols [22]. Actually, the multitarget features and aspecific mechanisms are the rationale for the use of phytocomplexes in modern phytotherapy. The C. sativa leaf extract, at dilutions of 1 : 100 and 1 : 200, showed a scavenging activity on H 2 O 2 -induced LPO, restoring similar levels of MDA measured in the control samples. To visualize the protective activity of the extract, a deep analysis of the single sperm organelles was performed using TEM that is a useful method to explore the morphological variation of human sperm treated in vitro with different compounds [20,21]. In general, TEM is one of the methods to study the different organelles characterizing sperm defects that can influence the fertilizing potential [44,45]. In this research, we demonstrated that the C. sativa leaf extract diluted to 1 : 200 was powerful in protecting sperm against severe oxidative damage induced by H 2 O 2 . The extract was able to defend sperm membranes and the acrosomes, both structures particularly affected by H 2 O 2 treatment.

Conclusions
The leaf extract of Castanea sativa Mill. var. Bastarda Rossa (Mount Amiata, Italy) is particularly rich in polyphenols and flavonoids and shows scavenging properties against oxidative stress induced by H 2 O 2 to human ejaculated sperm. This extract could be used in protocols of gamete handling such as techniques of assisted reproduction and cryopreservation of semen, all conditions in which oxidative stress is exacerbated. We are aware that further studies are needed to confirm the antioxidant ability of the C. sativa leaf extract. Should such activity be corroborated, these studies could be extended to the field of farming animals, in which the cryopreservation procedures are particularly common.

Data Availability
The data used to support the findings of this study are included within the article.