*3.4. Cytotoxicity*

Table 4 and Figure 12 summarize the IC50 values for the cytotoxic e ffect of *M. indica* extracts on di fferent human carcinoma cells, namely AGS, HepG2, and SW620 cell lines, all related to the digestive tract. The development of these types of cancers has been associated to a lower consumption of vegetables and fruits [57], particularly 60% of stomach cancer and 43% of colon cancer are attributed to deficient consumption of vegetables [58]. For this reason, it is interesting to evaluate the cytotoxicity activity of fruit phytochemical-enriched extracts on these tumoral cells.

**Table 4.** Cytotoxicity of *M. indica* extracts towards gastric (AGS), colon (SW-620), and liver (Hep-G2) carcinoma cells as well as towards Vero non-tumoral cells.


1 Different superscript letters in the same column indicates that differences are significant at *p* < 0.05. 2 Different superscript signs in the same row indicates differences are significant at *p* < 0.05.

The IC50 values shown in Table 4 sugges<sup>t</sup> that a better cytotoxic effect is displayed by the extracts obtained from skin than the extracts obtained from flesh. This difference is statistically significant for all cell lines in the T. Atkins cultivar and for SW-620 cells in the Keitt cultivar. The better results of the skin extracts are in agreemen<sup>t</sup> with results previously described for other cultivars [35,59,60]. Also, publications that compared extracts from different parts of the mango highlight the potential of skin and kernel extracts in the cytotoxic activity of different cell lines [61,62].

**Figure 12.** Cytotoxicity dose–response curves of *M. indica* extracts on AGS, HepG2, and SW620 adenocarcinoma cell lines and Vero normal cell lines. Results are presented as mean ± SD of three independent experiments. Mg-TA-f (T. Atkins cultivar flesh), Mg-K-f (Keitt cultivar flesh), Mg-TA-s (T. Atkins cultivar skin), and Mg-K-s (Keitt cultivar skin).

As shown in Table 4, in all assessed samples, the comparison of the cytotoxic effect between the different types of carcinoma cell lines showed better IC50 values against the gastric cells (AGS) than the hepatic (HepG2) and colon (SW-620) cell lines. Few reports of cytotoxic effects are available for gastric adenocarcinoma cell lines; for instance, for skin extracts of the Irwi mango cultivar, a dose-dependent effect from 125 to 1000 μg/mL was described on AGS cells [35] and a low cytotoxic effect on Kato-III cells using an ethanolic leaf extract of Okrong mango (IC50 > 200 μg/mL) [63]. Our results show better cytotoxic activity in ranges of 15 to 500 μg/mL, with an IC50 of 138 ± 8 μg/mL for Tommy Atkins skin and 197 ± 16 μg/mL for Keith skin cultivars.

Table 4 also shows a significantly better cytotoxic effect on the hepatocellular carcinoma cells (HepG2) for the T. Atkins cultivar compared to Keitt mango. In fact, the IC50 value assessed for T. Atkins skin is 164 ± 13 μg/mL, which is similar to results reported for the Irwin cultivar, which caused a decrease of 50% viability in concentrations between 125 and 250 μg/mL when incubated with HepG2 cells [35]. However, skin extracts of another mango species, *M. pajang*, showed better cytotoxic activity for HepG2 (IC50 = 36.5 μg/mL) than *M. indica* varieties [61].

On the other hand, the cytotoxic effect of *M. indica* cultivar skin extracts on colon carcinoma (SW-620) shows an IC50 of 175 ± 7 μg/mL for T. Atkins skin and 223 ± 24 μg/mL for the Keitt cultivar (Table 4). Previous reports for other varieties of *M. indica* cited IC50 values over 200 μg/mL for pulp extract [64] in similar colon carcinoma cell lines. In addition to the cytotoxic effect, the extracts used

in this study showed a significant difference (*p* < 0.05) between the IC50 of the control non-tumoral cell line (Vero) and the tumoral cell lines. This kind of selectivity is a desirable characteristic for any compound with chemotherapy potential [65,66].

Finally, the results of the correlation analysis of cytotoxic activities (Table 4) for each cell line with the abundance of polyphenolic compounds in each sample (Table 2) show a significant negative correlation between cytotoxic effects and the number of polyphenols for all cell lines, AGS (*r* = −0.984), HepG2 (*r* = −0.974), and SW620 (*r* = 0.983), at *p* < 0.05. In fact, T. Atkins skin constitutes the sample with more polyphenolic compounds and with the best cytotoxic activity, followed by the skin of the Keitt cultivar. Specific correlations between the cytotoxic activity and the presence of each type of polyphenolic compounds identified were calculated and it was observed that for AGS and SW 620 cell lines, the cytotoxic activity showed a particularly high significant negative correlation with gallotannins (*r* = −0.977 and *r* = −0.940, respectively), followed by a significant negative correlation of the SW620 cytotoxicity results with xanthonoids (*r* = −0.880). In turn, cytotoxic effects on the HepG2 cell line had the best significant negative correlation with xanthonoids (*r* = −0.921). These specific correlation values between the types of phenolic compounds and cytotoxic activity sugges<sup>t</sup> that gallotannins and xanthonoids play an important role in the toxicity against cancer cells. In fact, previous reports sugges<sup>t</sup> that these two classes of compounds seem to be strong determinants of the anti-tumor activity of mango extracts [14].

One of the main xanthonoids present in the skin extracts of our study is mangiferin and its isomeric forms (Table 2). These compounds have been described in the literature as promising anticancer polyphenols [11,67,68]. The ability of mangiferin to inhibit cancer cells is achieved through several molecular targets, however, one of the important mechanisms is associated with induction of apoptosis [68,69]. Although, besides apoptosis induction, other mechanisms of cell cytotoxicity have been postulated for mangiferin [11], such as cell cycle arrest [70] and a decrease in matrix metalloproteinase activities and reversal of the epithelial–mesenchymal transition [71].

Concerning gallates or gallotannins identified in mango extracts, in vitro studies have shown strong cytotoxic activity; for instance, inhibitions of 55% to 75% in the proliferation of breast, liver, and leukemia cancer cell lines treated with 40 to 80 μg/mL of extracts from Chinese cultivars [6]. The main compounds detected in these mango extracts correspond to pentagalloyl hexose to nonagalloyl hexose isomers. Also, these authors tested penta-galloyl hexoside and gallic acid to treat the same cell lines and confirm the potential role of these compounds as antiproliferatives. Another study on Keitt extract containing galloyl hexosides ranging from pentagalloyl hexose to nonagalloyl hexose inhibited 90% of breast cancer cell lines, with a concentration of 10 μg/mL evidencing strong activity [19].

Table 2 shows that skin extracts from mango contain a high number of gallotannins with different degrees of polymerization, ranging from monogalloyl hexose to undeca-galloyl hexoses. Studies of gallotannins from red maple species in colon and breast cancer cells showed an association between the higher number of galloyl groups in the gallotannins and better anticancer activity [22]. Particularly, the penta galloyl glucoside from fruits, such as maple, gallnuts, and oak, and medicinal herbs (*Galla rhois, Rhus chineensis, Paeonia su*ff*ruticosa*) has been widely associated to anticancer effects in prostate, breast, glioma, hepatocellular, and colorectal carcinoma [72–74]. The suggested mechanisms associated to the cytotoxicity involve induction of apoptosis through an increase of Bax/Bcl-2 protein levels, cell cycle arrest in S-phase, and the inhibition of NF-κB activation, with the consequent downregulation of inflammatory cytokines [73].

Even though our results and the cited previous reports sugges<sup>t</sup> that gallates and xanthonoids seem to be strong determinants of the anti-tumor activity, the mechanism of cell cytotoxicity has to be determined and the target molecules of the *M. indica* extract remain to be identified. Also, despite our results indicating a correlation with the presence of xanthonoids and gallates and the cytotoxic activity in tumoral cell lines, these compounds are probably not the only factors responsible for the observed biological effects. The contribution of other components present in the extracts should be clarified

because reports in the literature of similar anti-cancer properties are available for phenolic acids and flavonoids [75,76] and hydroxybenzophenones [77].
