Data for induction of cytotoxic response by natural and novel quercetin glycosides

The flavonoids quercetin, and its natural glycosides isoquercetin and rutin, are phytochemicals commonly consumed in plant-derived foods and used as a food beverage additive. Semi-synthetic maltooligosyl isoquercetin, monoglucosyl rutin and maltooligosyl rutin were developed by synthetic glycosylation to improve their water solubility for food and other applications. Using a system of Chinese hamster ovary (CHO) cells, this study examined the differences in cytotoxic responses induced by short and continuous exposure of natural and synthetic flavonoids. By assessing cell viability after short term exposure and clonogenicity with continuous exposure under various flavonoids, quercetin aglycone is confirmed to be the most cytotoxic flavonoids, and heavily glucosylated maltooligosyl rutin was the least cytotoxic. The other heavily glucosylated maltooligosyl isoquercetin showed intermediate cytotoxicity and similar toxicity as isoquercetin.


a b s t r a c t
The flavonoids quercetin, and its natural glycosides isoquercetin and rutin, are phytochemicals commonly consumed in plantderived foods and used as a food beverage additive. Semisynthetic maltooligosyl isoquercetin, monoglucosyl rutin and maltooligosyl rutin were developed by synthetic glycosylation to improve their water solubility for food and other applications. Using a system of Chinese hamster ovary (CHO) cells, this study examined the differences in cytotoxic responses induced by short and continuous exposure of natural and synthetic flavonoids. By assessing cell viability after short term exposure and clonogenicity with continuous exposure under various flavonoids, quercetin aglycone is confirmed to be the most cytotoxic flavonoids, and heavily glucosylated maltooligosyl rutin was the least cytotoxic.

Continuous induction of cytotoxic responses by the natural flavonoids and semi-synthetic
flavonoids confirmed that the natural flavonoids elicit greater cytotoxic responses than semisynthetic flavonoids.
Quercetin aglycone induced the highest cellular toxicity in the two assays we tested. Maltooligosyl rutin exhibited the least cytotoxicity. Maltooligosyl isoquercetin did not show reduced cytotoxicity compared to isoquercetin.

Data
Cell viability assessment was based on quantitative analysis of trypan blue dye exclusion after 24 h of exposure to the flavonoids. Quercetin showed approximately 15% cell death in 165 mM concentration and approximately 57% cell death in 330 mM concentration (Fig. 1A). In contrast, cells treated with isoquercetin, rutin, monoglycosyl rutin, maltooligosyl isoquercetin and maltooligosyl rutin did not show drastic cell death at the same or higher concentration ( Fig. 1B-F). Comparison of the flavonoid treatments indicates that exposure to quercetin resulted in the most pronounced cytotoxicity.
During colony formation, treatments with flavonoids were added continuously. Higher concentrations of all flavonoids showed a loss of clonogenicity. Under quercetin treatment, 50% of the cells were killed at concentrations of 20 and 30 mM ( Fig. 2A). More than 50% of the cells were killed under treatments with isoquercetin, maltooligosyl isoquercetin, and rutin at an approximate concentration of 500 mM (Fig. 2B-D). Half of the cells were killed under monoglucosyl rutin treatment at approximately 700 mM (Fig. 2E). However, cells exposed to maltooligosyl rutin did not reach 50% cell death, even at the concentration of 910 mM (Fig. 2F). This further confirmed that quercetin is the most cytotoxic natural flavonoid and indicated that maltooligosyl rutin is the least cytotoxic.

Colony formation assay
Trypsinized CHO cells were plated on P-60 dishes to obtain approximately 100 colonies per dish. Cells were treated with various dosages of natural and semi-synthetic flavonoids during colony formation. After a 7-day incubation period, cells were washed in 0.9% (w/v) sodium chloride and fixed in 100% ethanol, then stained with 0.1% (w/v) crystal violet dye (Sigma). Macroscopic colonies containing more than 50 cells (i.e., those formed from seeded cells that did not undergo a mitotic cell death following flavonoid exposure) were marked as survivors [4]. All studies were conduced as independent experiments for a minimum of three replicates for each endpoint.

Trypan blue dye exclusion assay
Cells were exposed to various concentrations of flavonoids for 24 h. After washing with PBS, trypan blue was added to the cells (final concentration was 0.004%). The dye exclusion test was carried out under Zeiss Axiovert S100 inverted microscope (Carl Zeiss, Jena, Germany) [5]. Blue stained cells were counted as dead cells. At least 200 cells were scored per data point. All studies were conducted as independent experiments for a minimum of three replicates for each endpoint.