Sinergia fra composti fitochimici estratti dalla bietola verde e rossa

The red beet contains a considerable amount of starch, together with organic acids, betalains, flavonoids, phenolic acids, minerals and vitamins. Concerning the betalains, their concentration is about 1%, depending on the cultivar. The aim of this study was to improve the conditions for chromatographic separation of betalains into betaxanthins (BX) and betacyanins (BC) and to investigate their biological activities towards cancer cell lines. We performed the betalain purification through a solid liquid extraction on the grated pulp, then a liquid-liquid extraction was applied to eliminate less soluble flavonoids. The acqueous phase was concentrated and loaded on the ion exchange chromatographic column DEAE Fast Flow. The betalains bound to the matrix were eluted with a saline gradient. This gradient eluted three fractions: R1, R2, R3. The R1 was a yellow powder, absorbing at 470nm, indicating the presence of betaxanthins; the second fraction (R2) was a red-violet powder, absorbing at 540 nm and containing betacyanins. The R3 fractions was a yellow-orange powder, representing a mixture of phenolic acids and modified betalains, showing a maximal abs at 330 nm. To obtain a further purification and to eliminate the salts, we performed a gel filtration on a G-10 column chromatography. The column was eluted with distilled water. The G-10 column gave us three desalted fractions: R1, containing mostly betaxanthins; R2 with the betacyanins and R3 with a mixture of compounds. The cytotoxic activity of R1, R2, R3 and the molecular mechanisms modulated by these phytochemicals were evaluated on two cancer cell lines: T24 from bladder cancer and CaCo-2 from colon cancer. In the T24 cells, the cytotoxic activity was significant only for R2 fraction, with a reduction of the viability at 24, 48 and 72 hours. Interestingly, no cytotoxic effect was observed on normal epithelial NCTC 2544 cell line. In T24 cells, the betacyanins increased caspase 8 and 3 activities and down regulated the expression of the gene CTNNB1 (β-catenin), increasing apoptosis and inhibiting the proliferation rate of these cancer cells. In T24 cancer cells the combination of betalains with XVX resulted in a strong increase of their cytotoxic activity, as XVX activated the intrinsic apoptotic pathway (through the upregulation of BAX and downregulation of BIRC 5), while betacyanins the extrinsic pathway. In CaCo-2 cell line, the cytotoxic activity was significant for R1 and R2 fractions at 24h treatment. Through the analysis of the cytotoxicity at 24h, an hormetic effect was observed. Both BX and BC can exerted their antioxidant activity inside CaCo-2 cells, as demonstrated through the DCFH-DA assay. The molecular mechanisms of the betalain action in CaCo-2 cells was based on a significant increse of the Bax/Bcl protein ratio, in particular with the BC, which also increased cleaved PARP1 and cleaved caspase 3 protein levels. At a molecular level, the BC downregulated COX-2 and IL-8 mRNA levels, thus acting as anti- inflammatory agents. Both the antiproliferative and the anti-inflammatory effects were remarkably increased when BX and BC were used in combination with vitexin-2O-xyloside (XVX) in CaCo-2 cancer cells. The antiproliferative effects of betalains were also tested on p53wt HepG2 hepatocarcinoma cells, p53 mut HuH7 liver cancer cells and rat glioblastoma C6 cells. Betalains showed a slight cytotoxic effect against HepG2 cells and this cytotoxicity increased when BX and BC were used in combination with XVX. Otherwise, betalains showed no cytotoxic effects on C6 and HuH7 cells. Interestingly, BX and BC exerted an antioxidant activity in HuH7 cells through the increase of transcriptional activity of Nrf2, the transcription factor that regulates the cellular antioxidant mechanisms. The interest regarding betalains is very high for human health, as the red beet powder is used as the unique red dye (E 162)in foods and the purified betalains, added in food preparations, may work as anti-inflammatory and chemopreventive age