## 4 Discussion
The antioxidant activity of polyphenols could be essential but not sufficient for chemoprevention [60], and there is evidence that naturally occurring antioxidants elicit different redox responses according to a dose-response mechanism and the intracellular redox state [61; 62; 63]. Like many other species, the chestnut phenolic compounds have been correlated with anticancer activity. Unfortunately, studies on the anticancer properties of chestnut are still limited to a few articles: one tumor (DU 145) and one normal prostate epithelial (PNT2) cell line were exposed to increasing concentration of shell polyphenols extracts, thus showing an increase of apoptotic cells after 48 and 72 h treatments [12]; anticancer activity vs. prostate cancer cells (DU 145 and PC3) was attributed to a quinoline-pyrrolidine alkaloid present in chestnut honey, and the inhibition of clonogenic activity was congruent with apoptotic events [39]. Apoptotic-induced effects were also observed on neuroblastoma cells (SH-SY5Y) when exposed to increasing concentration extracts obtained by bark of _Castanea sativa_ containing high amounts of vescalagin, castalagin gallic and ellagic acid [40]. In our work, normal cells and four cancer cell lines, SW48, RKO, MCF7 and B16F10, were exposed to cold and hot water pericarp extracts of four Sardinian accessions and of one marron cultivar. The temperature and different extraction times significantly influenced the growth of cells, both normal and tumor. The fibroblast viability was significantly reduced, at least by 20%, by treatments with cold extracts starting from 100 \(\upmu\)g mL\({}^{-1}\) of MURG and COEV, 50 \(\upmu\)g mL\({}^{-1}\) of LOCG and ILDP, and even 10 \(\upmu\)g mL\({}^{-1}\) of MARRV. According to [PERSON] et al. [64], a 20% reduction in viability is the threshold to consider a treatment as being effective. Such a reduction, with hot extracts, was observed only with doses greater than 250 \(\upmu\)g mL\({}^{-1}\), regardless of the accession or cultivar to which they belong, and were confirmed by GI50 values obtained by nonlinear regression analysis. This clearly indicates that the pool of molecules extracted at 95 \({}^{\circ}\)C exerts a cytotoxic effect significantly lower than that of cold extracts. Even on cancer cells, the most evident cytotoxic effects were observed when the cells were treated with cold extracts. The viability of metastatic colon cancer cells, SW48 and RKO, was strongly reduced by 100 \(\upmu\)g mL\({}^{-1}\) (if not even 50 \(\upmu\)g mL\({}^{-1}\) of LOCG) of all the Sardinian accessions extracts, while MARRV had no effect; also in this case, the cytotoxic action of the hot extracts was unquestionably lower, and only at the highest doses. Furthermore, it was observed that the GI50 values calculated on colon cancer cells in this work, are lower than those reported for infusions and decoctions of _Castanea sativa_ flowers on HCT15 (colon carcinoma) cells [65]. The activity against colon cancer cells of chestnut extracts was also investigated by other research groups. The capacity of inner shell extracts of Japanese chestnut (_Castanea crenata_ [PERSON], 1846), to inhibit the viability of LoVo and HT29, two human colon adenocarcinoma cell lines, was explored [20]: the water extracts (24 h at 60 \({}^{\circ}\)C), containing 2.78 mg g\({}^{-1}\) of gallic acid (higher than ours) and 1.07 mg g\({}^{-1}\) of ellagic acid (lower than ours), induced a viability reduction, varying between 20 and 60%, starting from 125 \(\upmu\)g mL\({}^{-1}\), a result comparable to those obtained by our group on RKO and SW48. On the other hand, the bioactive compounds of shells of Chinese chestnut (_Castanea molissima_ [PERSON]) seemed to have a moderate anti-cancer activity on human COLO 320 DM colon cancer cells, but results cannot be compared with ours since a 90:10 (\(v/v\)) ethanol-water solution was used as extractant [66].