Abatement of the Stimulatory Effect of Copper Nanoparticles Supported on Titania on Ovarian Cell Functions by Some Plants and Phytochemicals

The application of nanoparticles has experienced a vertiginous growth, but their interaction with food and medicinal plants in organisms, especially in the control of reproduction, remains unresolved. We examined the influence of copper nanoparticles supported on titania (CuNPs/TiO2), plant extracts (buckwheat (Fagopyrum esculentum) and vitex (Vitex agnus-castus)), phytochemicals (rutin and apigenin), and their combination with CuNPs/TiO2 on ovarian cell functions, using cultured porcine ovarian granulosa cells. Cell viability, proliferation (PCNA accumulation), apoptosis (accumulation of bax), and hormones release (progesterone, testosterone, and 17β-estradiol) were analyzed by the Trypan blue test, quantitative immunocytochemistry, and ELISA, respectively. CuNPs/TiO2 increased cell viability, proliferation, apoptosis, and testosterone but not progesterone release, and reduced the 17β-estradiol output. Plant extracts and components have similar stimulatory action on ovarian cell functions as CuNPs/TiO2, but abated the majority of the CuNPs/TiO2 effects. This study concludes that (1) CuNPs/TiO2 can directly stimulate ovarian cell functions, promoting ovarian cell proliferation, apoptosis, turnover, viability, and steroid hormones release; (2) the plants buckwheat and vitex, as well as rutin and apigenin, can promote some of these ovarian functions too; and (3) these plant additives mitigate the CuNPs/TiO2’s activity, something that must be considered when applied together.


Introduction
Nanotechnology and the applications of metal nanoparticles (i.e., particles with a diameter < 100 nm) have rapidly developed in recent years [1][2][3][4]. In particular, nanoparticles of the relatively cheap and abundant copper (CuNPs) have found multiple applications in diverse areas, including catalysis [5] and materials science [6,7]. However, CuNPs have also been exploited for disinfection purposes because of their effective antimicrobial properties, in medicinal chemistry or as farm animal food additives [8][9][10]. Nevertheless, copper nanoparticles can adversely influence various biological processes [11][12][13] and produce some toxicological effects on individual organisms [14].
Assays conducted on rodents proved the ability of both bulk copper and CuNPs to diminish the levels of blood gonadal and gonadotropin steroid hormones to cause degenerative alterations in gonads, ovarian follicular atresia, and to inhibit gamete and embryogenesis [15][16][17]. This detrimental behavior the plant/phytochemical. For this purpose, we compared the influence of CuNPs/TiO 2 (0, 1, 10, or 100 µg/mL), buckwheat, vitex, rutin, and apigenin (10 µg/mL) when given individually and when CuNPs/TiO 2 was combined with plant additives. We compared the activity of these treatments on cultured porcine ovarian granulosa cells by studying cell viability, the accumulation of bax (a cytoplasmic apoptosis marker) [53,54], PCNA (a proliferation marker) [55], and the release of the steroid hormones testosterone, progesterone, and 17β-estradiol, the regulators and markers of ovarian cell functions [56]. This survey reveals that CuNPs/TiO 2 can boost some of the aforementioned ovarian cell functions, as buckwheat, vitex, rutin, and apigenin also do. However, the activity CuNPs/TiO 2 is significantly depleted in the presence of these plant additives.

Preparation of Copper Nanoparticles Supported on Titania (CuNPs/TiO 2 )
The methodology of Alonso's group [21] was applied for the synthesis of CuNPs/TiO 2 . In this protocol, a suspension was prepared in a Schlenk flask containing lithium powder (14 mg, 2.0 mmol, Medalchemy S.L.) and 4,4 -di-tert-butylbiphenyl (27 mg, 0.1 mmol; DTBB, Sigma-Aldrich, St. Louis, MO, USA) in dry tetrahydrofuran as a solvent (THF, 20 mL) under argon at room temperature. Anhydrous CuCl 2 (134 mg, 1.0 mmol; 97%, Sigma-Aldrich, St. Louis, MO, USA) was added to this suspension. The initially formed dark-green reaction mixture changed to black, as a result of the formation of CuNPs. Then, additional dry THF (18 mL) and TiO 2 (1.28 g, titania anatase nanopowder, Alfa Aesar, Ward Hill, MA, USA) were added to the above suspension. The reaction mixture was stirred at room temperature for 1 h, followed by filtration and drying under air.

Isolation and Culture of Granulosa Cells
The granulosa cells were isolated from the ovaries of non-cycling pubertal gilts (180 days age, approximately), and were processed and cultured as previously described [57][58][59][60][61]. Briefly, the collected granulosa cells (at 10 6 cells/mL concentration) were precultured in sterile Dulbecco's modified Eagle medium (DMEM)/F12 1:1, containing 10% fetal calf serum (both from BioWhittakerTM, Verviers, Belgium) and 1% antibiotic-antimycotic solution (Sigma-Aldrich, St. Louis, MO, USA) in 16-well (200 µL/well) chamber slides (Nunc Inc., International, Naperville, IL, USA) during 3-4 days. Buckwheat (Fagopyrum esculentum), vitex (Vitex agnus-castus; both from F-DENTAL Hodonín s.r.o., Hodonín, Czech Republic), rutin (WallMark, a.s., Třinec, Czech Republic), and apigenin (Merck KGaA, Darmstadt, Germany) were commercially available. The plants were minced for 2 min in a coffee mill. Thereafter, either the plant powder or the plant components apigenin and rutin were dissolved in 50 µL of DMSO in order to make stock solutions of 1 mg/mL. These stock solutions were dissolved in the culture medium immediately before their addition to the cells, in such a way that the final concentration of DMSO did not exceed 0.001%. Previous studies revealed no substantial effects of 0.001% DMSO on ovarian cell function and viability. Controls included ovarian cells cultured in the incubation medium (with 0.001% DMSO) without treatment and the medium incubated without cells (blank control).
The original medium was replaced with one of the same composition without and with CuNPs/TiO 2 , at the concentrations of 0, 1, 10, or 100 µg/mL, with buckwheat, vitex, rutin, and apigenin, each at 10 µg/mL. In addition, CuNPs/TiO 2 (0, 1, 10, or 100 µg/mL) was combined with each of the plant additives listed above (10 µg/mL). In this case, the plant additive was first added to the cell culture and, after 10-15 min, CuNPs/TiO 2 was added to the resulting medium. These doses correspond to those of CuNPs that can be ingested and that have been applied in previous animal experiments, both in vivo and in vitro [20,[58][59][60][61]. The dose of the plant additives was selected on the basis of the doses used in the corresponding previous in vitro experiments [41,43,45,49,50]. These substances were dissolved in the culture medium just before their addition to the cells. The cell and the incubation medium were analyzed after two days of culture.

Cell Viability Test
The Trypan blue exclusion test was used to evaluate the cell viability, according to Strober [62]. Succinctly, removal of the medium from the culture plates was done after incubating the granulosa cells. Then, the Trypan blue staining (Sigma-Aldrich, Hamburg, Germany) was applied to the cell monolayer for 15 min. After removal of this dye, the plates were subjected to washing (twice with the physiological solution) and to microscopic inspection (magnification: 400×). The ratio of dead (stained) cells to the total cell count was calculated.

Immunocytochemical Analysis of Proliferation and Apoptosis Markers
Immunocytochemistry was used to detect the presence of PCNA and bax in the cells, as described previously [19,20,[57][58][59][60][61], by means of primary monoclonal antibodies against PCNA and bax (dilution 1:500; from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), secondary swine antibodies against mouse IgG labeled with horseradish peroxidase (dilution 1:1000; Servac, Prague, Czech Republic) and visualized by DAB-substrate staining (Roche Diagnostics GmbH, Manheim, Germany). In some cases, the assay was validated by these primary antibodies and secondary polyclonal goat antibodies against mouse IgGs, labeled with fluorescein isothiocyanate (FITC, dilution 1:1000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA; Figure 1). The negative controls were performed with cells processed without the primary or secondary antibody. The cells were inspected with the aid of a light and a fluorescence microscope (Leica GmbH, Wetzlar, Germany). The cells showing a signal larger than that of the levels of the background negative controls were considered positive. The percentage of cells containing a visible signal-marker of PCNA and bax was calculated relative to the total cell number.

Cell Viability Test
The Trypan blue exclusion test was used to evaluate the cell viability, according to Strober [62]. Succinctly, removal of the medium from the culture plates was done after incubating the granulosa cells. Then, the Trypan blue staining (Sigma-Aldrich, Hamburg, Germany) was applied to the cell monolayer for 15 min. After removal of this dye, the plates were subjected to washing (twice with the physiological solution) and to microscopic inspection (magnification: 400×). The ratio of dead (stained) cells to the total cell count was calculated.

Immunocytochemical Analysis of Proliferation and Apoptosis Markers
Immunocytochemistry was used to detect the presence of PCNA and bax in the cells, as described previously [19,20,[57][58][59][60][61], by means of primary monoclonal antibodies against PCNA and bax (dilution 1:500; from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), secondary swine antibodies against mouse IgG labeled with horseradish peroxidase (dilution 1:1000; Servac, Prague, Czech Republic) and visualized by DAB-substrate staining (Roche Diagnostics GmbH, Manheim, Germany). In some cases, the assay was validated by these primary antibodies and secondary polyclonal goat antibodies against mouse IgGs, labeled with fluorescein isothiocyanate (FITC, dilution 1:1000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA; Figure 1). The negative controls were performed with cells processed without the primary or secondary antibody. The cells were inspected with the aid of a light and a fluorescence microscope (Leica GmbH, Wetzlar, Germany). The cells showing a signal larger than that of the levels of the background negative controls were considered positive. The percentage of cells containing a visible signal-marker of PCNA and bax was calculated relative to the total cell number.

Immunoassay of Hormones
Progesterone, testosterone, and 17β-estradiol concentrations were determined from 25 µL aliquots of the incubation medium through the enzyme-linked immunosorbent assay (ELISA), utilizing ELISA's kits (LDN Immunoassays and Services, Nodhorn, Germany), following the instructions of the manufacturer. The details of the assay were reported previously [20]. Validation of all the assays was accomplished by dilution tests of culture medium samples.

Statistical Analysis
Each experiment was repeated thrice using ovaries from different animals (10-15 ovaries per experiment). In some experiments, all the treatments were tested at once, in other experiments the effects of only part of the additives were investigated (for example, CuNPs/TiO2 alone and in combination with rutin or apigenin). The presented data are the summarized results obtained from, at least, three independent experiments. Each group of experiments was represented by four culture wells. Immunocytochemical and viability tests were performed on, at least, 1000 cells per group. By using ELISA, blank control values were subtracted from those determined in the cell-conditioned medium in order to exclude any non-specific background (<13% of the total values). Secretion rates

Immunoassay of Hormones
Progesterone, testosterone, and 17β-estradiol concentrations were determined from 25 µL aliquots of the incubation medium through the enzyme-linked immunosorbent assay (ELISA), utilizing ELISA's kits (LDN Immunoassays and Services, Nodhorn, Germany), following the instructions of the manufacturer. The details of the assay were reported previously [20]. Validation of all the assays was accomplished by dilution tests of culture medium samples.

Statistical Analysis
Each experiment was repeated thrice using ovaries from different animals (10-15 ovaries per experiment). In some experiments, all the treatments were tested at once, in other experiments the effects of only part of the additives were investigated (for example, CuNPs/TiO 2 alone and in combination with rutin or apigenin). The presented data are the summarized results obtained from, at least, three independent experiments. Each group of experiments was represented by four culture wells. Immunocytochemical and viability tests were performed on, at least, 1000 cells per group. By using ELISA, blank control values were subtracted from those determined in the cell-conditioned Nanomaterials 2020, 10, 1859 5 of 14 medium in order to exclude any non-specific background (<13% of the total values). Secretion rates were calculated for 10 6 cells/day. Differences between groups were evaluated using the Shapiro-Wilk's normality and Student's t-tests and Sigma Plot 11.0 (Systat Software, GmbH, Erkhart, Germany). The data was expressed as the mean ± standard error of mean (SEM). Differences were considered statistically significant at p ≤ 0.05.

Results
The solid composed of copper nanoparticles on titania (CuNPs/TiO 2 ) was characterized by transmission electron microscopy (TEM), showing well dispersed spherical CuNPs of an 0.98 ± 0.42 nm average size ( Figure 2). The copper loading was determined to be 1.9 wt% by inductively coupled plasma optical emission spectroscopy (ICP-OES). The oxidation state of the copper species was analyzed by X-ray photoelectron spectroscopy (XPS), denoting the presence of both Cu(I) and Cu(II) oxide because of the exposure of the nanoparticles to air [20]. were calculated for 10 6 cells/day. Differences between groups were evaluated using the Shapiro-Wilk's normality and Student's t-tests and Sigma Plot 11.0 (Systat Software, GmbH, Erkhart, Germany). The data was expressed as the mean ± standard error of mean (SEM). Differences were considered statistically significant at p ≤ 0.05.

Results
The solid composed of copper nanoparticles on titania (CuNPs/TiO2) was characterized by transmission electron microscopy (TEM), showing well dispersed spherical CuNPs of an 0.98 ± 0.42 nm average size ( Figure 2). The copper loading was determined to be 1.9 wt% by inductively coupled plasma optical emission spectroscopy (ICP-OES). The oxidation state of the copper species was analyzed by X-ray photoelectron spectroscopy (XPS), denoting the presence of both Cu(I) and Cu(II) oxide because of the exposure of the nanoparticles to air [20]. The cells in all the groups were viable, contained markers of apoptosis (bax) and proliferation (PCNA), and secreted substantial amounts of the steroids progesterone, testosterone, and 17β-estradiol. The addition of CuNPs/TiO2, buckwheat, vitex, rutin, and apigenin was able to alter these parameters.
Buckwheat, when added alone, enhanced cell viability ( Figure 3A, see CuNPs/TiO2 at the dose of 0 µg/mL) and accumulation of bax ( Figure 3C), whereas it lowered the testosterone release ( Figure  3E). No significant buckwheat influence on PCNA ( Figure 3B), progesterone ( Figure 3D), and 17β-estradiol ( Figure 3F) was observed. The influence of CuNPs/TiO2 on cell viability ( Figure 3A), PCNA/proliferation ( Figure 3B), bax/apoptosis ( Figure 3C), the release of testosterone ( Figure 3E), and 17β-estradiol ( Figure 3F) was significantly lower in the presence of buckwheat than in its absence. Moreover, buckwheat not only prevented but also even inverted the action of CuNPs/TiO2 on bax-CuNPs/TiO2 (at 100 µg/mL). The presence of buckwheat did not promote but inhibited bax accumulation ( Figure 3C). The presence of buckwheat did not substantially modify the CuNPs/TiO2 (1 or 10 µg/mL) effect on the progesterone release, but CuNPs/TiO2 added at a dose of 100 µg/mL failed to promote progesterone output in the presence of buckwheat ( Figure 3D). The cells in all the groups were viable, contained markers of apoptosis (bax) and proliferation (PCNA), and secreted substantial amounts of the steroids progesterone, testosterone, and 17β-estradiol. The addition of CuNPs/TiO 2 , buckwheat, vitex, rutin, and apigenin was able to alter these parameters.
Buckwheat, when added alone, enhanced cell viability ( Figure 3A, see CuNPs/TiO 2 at the dose of 0 µg/mL) and accumulation of bax ( Figure 3C), whereas it lowered the testosterone release ( Figure 3E). No significant buckwheat influence on PCNA ( Figure 3B), progesterone ( Figure 3D), and 17β-estradiol ( Figure 3F) was observed. The influence of CuNPs/TiO 2 on cell viability ( Figure 3A), PCNA/proliferation ( Figure 3B), bax/apoptosis ( Figure 3C), the release of testosterone ( Figure 3E), and 17β-estradiol ( Figure 3F) was significantly lower in the presence of buckwheat than in its absence. Moreover, buckwheat not only prevented but also even inverted the action of CuNPs/TiO 2 on bax-CuNPs/TiO 2 (at 100 µg/mL). The presence of buckwheat did not promote but inhibited bax accumulation ( Figure 3C). The presence of buckwheat did not substantially modify the CuNPs/TiO 2 (1 or 10 µg/mL) effect on the progesterone release, but CuNPs/TiO 2 added at a dose of 100 µg/mL failed to promote progesterone output in the presence of buckwheat ( Figure 3D). Vitex, when given alone, increased cell viability ( Figure 4A), PCNA ( Figure 4B) and bax ( Figure  4C) accumulation, and the release of 17β-estradiol ( Figure 4F) but not of progesterone ( Figure 4D) or testosterone ( Figure 4E). Furthermore, vitex prevented the stimulatory action of CuNPs/TiO2 on cell viability ( Figure 4A), the accumulation of PCNA ( Figure 4B) and bax ( Figure 4C), and on the testosterone release ( Figure 4E). Moreover, it changed the stimulatory action of CuNPs/TiO2 on bax ( Figure 4C) and 17β-estradiol ( Figure 4F) to an inhibitory one. Vitex did not modify the CuNPs/TiO2 influence on the progesterone output ( Figure 4D).   Figure  4C) accumulation, and the release of 17β-estradiol ( Figure 4F) but not of progesterone ( Figure 4D) or testosterone ( Figure 4E). Furthermore, vitex prevented the stimulatory action of CuNPs/TiO2 on cell viability ( Figure 4A), the accumulation of PCNA ( Figure 4B) and bax ( Figure 4C), and on the testosterone release ( Figure 4E). Moreover, it changed the stimulatory action of CuNPs/TiO2 on bax ( Figure 4C) and 17β-estradiol ( Figure 4F) to an inhibitory one. Vitex did not modify the CuNPs/TiO2 influence on the progesterone output ( Figure 4D). Apigenin promoted cell viability ( Figure 6A), PCNA accumulation ( Figure 6B), and the secretion of testosterone ( Figure 6E) and 17β-estradiol ( Figure 6F), whereas it shortened the progesterone release ( Figure 6D) and had no effect on the accumulation of bax ( Figure 6C). In the presence of apigenin, CuNPs/TiO2 lost its ability to promote cell viability ( Figure 6A), accumulation of PCNA ( Figure 6B) and the release of testosterone ( Figure 6E), but not to boost the accumulation of bax ( Figure 6C) or the release of 17β-estradiol ( Figure 6F). Moreover, CuNPs/TiO2 could inhibit the progesterone release in the presence of apigenin ( Figure 6D).

Discussion
The formation of a monolayer, the high cell viability, the presence of intracellular markers of proliferation and apoptosis and the production of steroid hormones denote that the porcine granulosa cells tested were in good condition and adequate for the analyses and testing of the effects of CuNPs/TiO2, plant extracts and phytochemicals. The character of the changes induced by these Vitex, when given alone, increased cell viability ( Figure 4A), PCNA ( Figure 4B) and bax ( Figure 4C) accumulation, and the release of 17β-estradiol ( Figure 4F) but not of progesterone ( Figure 4D) or testosterone ( Figure 4E). Furthermore, vitex prevented the stimulatory action of CuNPs/TiO 2 on cell viability (Figure 4A), the accumulation of PCNA ( Figure 4B) and bax ( Figure 4C), and on the testosterone release ( Figure 4E). Moreover, it changed the stimulatory action of CuNPs/TiO 2 on bax ( Figure 4C) and 17β-estradiol ( Figure 4F) to an inhibitory one. Vitex did not modify the CuNPs/TiO 2 influence on the progesterone output ( Figure 4D).
Apigenin promoted cell viability ( Figure 6A), PCNA accumulation ( Figure 6B), and the secretion of testosterone ( Figure 6E) and 17β-estradiol ( Figure 6F), whereas it shortened the progesterone release ( Figure 6D) and had no effect on the accumulation of bax ( Figure 6C). In the presence of apigenin, CuNPs/TiO 2 lost its ability to promote cell viability ( Figure 6A), accumulation of PCNA ( Figure 6B) and the release of testosterone ( Figure 6E), but not to boost the accumulation of bax ( Figure 6C) or the release of 17β-estradiol ( Figure 6F). Moreover, CuNPs/TiO 2 could inhibit the progesterone release in the presence of apigenin ( Figure 6D).

Discussion
The formation of a monolayer, the high cell viability, the presence of intracellular markers of proliferation and apoptosis and the production of steroid hormones denote that the porcine granulosa cells tested were in good condition and adequate for the analyses and testing of the effects of CuNPs/TiO 2 , plant extracts and phytochemicals. The character of the changes induced by these additives and their combinations are summarized in Table 1 The performed studies demonstrated the stimulatory influence of CuNPs/TiO 2 on five out of the six measured ovarian cell functions; only one parameter remained unchanged. These observations suggest the absence of toxicity of copper nanoparticles supported on titania, in contrast to a variety of other copper nanoparticles [11,12,14]. Moreover, these results are in line with our previous observation about the stimulatory activity of this type of nanoparticles on ovarian cell functions. The mechanism of this effect and the functional interrelationships between the measured parameters require further elucidation. Nevertheless, CuNPs/TiO 2 induced an increase of both, proliferation and cytoplasmic apoptosis, suggesting the ability of these nanoparticles to promote ovarian cell turnover. In addition to this, CuNPs/TiO 2 also increased cell viability, a result that could be rationalized by the dominance of cell proliferation over cell apoptosis. Intensified apoptosis could be a result of the augmented release of testosterone, a promoter of ovarian follicular atresia and cytoplasmic apoptosis. Finally, the growth in proliferation might be due to a larger production of 17β-estradiol, which is a known promoter of cell proliferation and viability [56]. In any case, it must be taken into account that cell viability can be affected not only by cytoplasmic apoptosis (induced by bax [53,54]) but, alternatively, by nuclear apoptosis associated with nuclear DNA fragmentation [63,64], which has not been analyzed herein. It has been demonstrated that some regulators can induce the opposite changes in cytoplasmic and nuclear apoptosis in porcine granulosa cells [57]. Therefore, it cannot be completely excluded that CuNPs/TiO 2 might suppress nuclear apoptosis, which, together with increased proliferation, could boost cell viability. If the stimulatory action of CuNPs/TiO 2 on the ovary occurred in vivo too, the potential application of CuNPs/TiO 2 as a safe replacement of toxic CuNPs and a novel biostimulating agent of reproductive processes in animals and humans cannot be disregarded. The performed studies demonstrate the direct influence of some plant extracts and their phytochemicals on ovarian cell functions. The ability of buckwheat to enhance viability and apoptosis of cultured cells is the first evidence on the direct action of buckwheat on ovarian functions. The mechanisms of these buckwheat effects remain unknown. The stimulatory action of vitex on ovarian cell viability, proliferation, apoptosis, and 17β-estradiol release is concordant with previous indications on its ability to increase ovarian cell apoptosis [41,42] and steroidogenesis [36][37][38][39][40]. Furthermore, there is the first evidence on the capability of vitex to promote ovarian cell proliferation, resulting in cell turnover and viability. These effects explain the stimulatory and therapeutic action of vitex on some ovarian functions reported previously [36][37][38][39][40].
The tested phytochemicals exhibit stimulatory activity on some ovarian cell functions similar to the stimulatory effects shown by the whole plant extracts. For example, in our experiments, rutin boosted cell viability and the release of testosterone and 17β-estradiol. It cannot be ruled out that the cell viability increase could be attributed to the increased release of the anti-atretic hormone 17β-estradiol [62], though the mechanism of rutin's role is worthy of further studies. The present results are the first evidence about the direct stimulatory activity of rutin on healthy ovarian cells. This activity is in agreement with the therapeutic effect of this polyphenol on an ovary that suffered from ischemia-reperfusion injury [44], restraint [45], and polycystic ovarian syndrome [47], reported previously.
Another polyphenolic phytochemical, apigenin, also manifested stimulatory behavior on several ovarian functions (viability, proliferation, testosterone, and 17β-estradiol release), albeit it suppressed the progesterone output. The promotion of cell viability by apigenin can be explained by its capacity to promote ovarian cell proliferation, but not apoptosis, i.e., to change the proliferation/apoptosis rate. This effect could be attributed to the increased secretion of 17β-estradiol, which is considered an inducer of ovarian cell proliferation. What is more, the increase of cell viability, proliferation and release of 17β-estradiol, and the decrease of progesterone release suggest that apigenin could be a natural stimulating agent of ovarian follicle survival, growth, and an inhibitor of follicular luteinization and atresia, which are under control of these hormones [56]. This is the first proof of the apigenin activity on healthy ovarian cells, which supports the potential benefits of apigenin-containing products for the promotion of animal and human reproductive processes. Furthermore, the changes induced by apigenin in healthy ovarian cells, observed in the present experiments, explain its ability to promote ovarian functions during polycystic ovarian syndrome [51] and ischemia-reperfusion injury [52]. Moreover, the ability of apigenin to induce apoptosis and to reduce the viability of cancer cells, observed previously [50], but not those in healthy ovarian cells (in our experiments), sustains a potential applicability of apigenin for the selective suppression of cancer and for the support of healthy cells in the ovary.
The similar activity found for some plant extracts and plant components on some ovarian cell parameters might indicate, in principle, that the plants tested could affect some ovarian functions because of the presence of the phytochemicals rutin and apigenin among their components. However, only a small proportion of their effects was found to be similar, probably because of the participation of other molecules or molecule complexes. The plants effect could not be explained by the presence of another ubiquitous polyphenolic phytochemical, quercetin, because its suppressive action on all the ovarian functions reported previously [58][59][60][61] is opposed to that observed for buckwheat and vitex in the present study. Therefore, it seems that rutin, apigenin, and quercetin cannot explain the action of buckwheat or vitex on ovarian cells, and that these polyphenols cannot replace the treatment with the whole plant extract.
Interestingly, the plants and the polyphenolic flavonoids tested were able not only to affect basic ovarian cell functions, but also to diminish the stimulatory action of CuNPs/TiO 2 on these functions. In particular, buckwheat prevented the action of CuNPs/TiO 2 on all measured indexes and vitex inhibited the activity of CuNPs/TiO 2 in five out of the six measured parameters, whereas rutin and apigenin did the same in four out of six parameters. None of the plant additives promoted any CuNPs/TiO 2 effect, which might indicate that the tested plant additives could oppose the action of CuNPs/TiO 2 . Based on our experience on CuNPs [23][24][25][26], we know that their catalytic activity in chemical reactions can be depleted or even completely blocked by their interaction with hydroxyl groups present in organic molecules. Taking into account that plant extracts, as well as rutin and apigenin, are rich in hydroxyl groups, because of the presence of carbohydrate and/or phenolic units, their binding to the surface of the CuNPs could passivate it, with the concomitant reduction of CuNPs' bioactivity. This behavior could have some health consequences and should be taken into account in view of some potential medical applications, avoiding their joint prescription. Moreover, it cannot be discarded that a daily diet containing these plants and/or their components could reduce the application efficiency of CuNPs/TiO 2 .
The performed experiments did not address all the possible questions concerning the mechanisms of action and interrelationships of the different ovarian regulators. Nevertheless, they show (1) that CuNPs/TiO 2 could directly stimulate ovarian cell functions; (2) that CuNPs/TiO 2 could positively affect ovarian cell proliferation, apoptosis, turnover, viability, and steroid hormones release; (3) that the plants buckwheat and vitex, and the phytochemicals rutin and apigenin, could promote some of these ovarian functions too; and (4) that these plant additives could not promote but abate the action of CuNPs/TiO 2 , what must be taken into account for their potential application. Therefore, given the ability of food and medicinal plants and their polyphenolic constituents to suppress the activity of CuNPs, to intake CuNPs and these plants or plant components jointly seems unadvisable concerning the ovarian functions. However, this interaction can be beneficial to prevent the potential noxious effect, when accidentally exposed to CuNPs, on other types of cells or organs.