In vitro antioxidant activity of phenolic-enriched extracts from Zhangping Narcissus tea cake and their inhibition on growth and metastatic capacity of 4T1 murine breast cancer cells

Abstract

Phenolics, as the main bioactive compounds in tea, have been suggested to have potential in the prevention of various human diseases. However, little is known about phenolics and their bioactivity in Zhangping Narcissue tea cake which is considered the most special kind of oolong tea. To unveil its bioactivity, three phenolic-enriched extracts were obtained from Zhangping Narcissue tea cake using ethyl acetate, n-butanol, and water. Their main chemical compositions and in vitro bioactivity were analyzed by high-performance liquid chromatography (HPLC) and ultra-performance liquid chromatography-mass spectrometry (UPLC-MS). The ethyl acetate fraction (ZEF) consisted of higher content of phenolics, flavonoids, procyanidins, and catechin monomers (including epigallocatechin gallate (EGCG), epicatechin gallate (ECG), and gallocatechin gallate (GCG)) than n-butanol fraction (ZBF) and water fraction (ZWF). ZEF exhibited the strongest antioxidant capacity in vitro due to its abundant bioactive compounds. This was validated by Pearson correlation and hierarchical clustering analyses. ZEF also showed a remarkable inhibition on the growth, migration, and invasion of 4T1 murine breast cancer cells.

Keywords: Oolong tea; Antioxidant activity; Metastasis; Cell cycle.

Fig. 1

HPLC chromatograms of catechin monomers and theaflavin in ZEF, ZBF, and ZWF (a) Standard mixture; (b–d) ZEF (b), ZBF (c), and ZWF (d) are extracts from Zhangping Narcissus tea cake by ethyl acetate, n-butanol, and water, respectively

Fig. 2

Antioxidant activity of ZEF, ZBF, ZWF, BHT, and EGCG and inhibition of 4T1 cell growth by ZEF, ZBF, and EGCG (a) DPPH scavenging activity; (b) ABTS scavenging capacity; (c) FRAP. ZEF, ZBF, and ZWF are extracted from Zhangping Narcissus tea cake by ethyl acetate, n-butanol, and water, respectively. BHT and EGCG are the positive controls. Error bars indicate the standard deviations (n=3)

Fig. 3

Hierarchical clustering of chemical constitution and antioxidant activity in ZEF, ZBF, and ZWF (a) and inhibition of growth by ZEF, ZBF, and EGCG in 4T1 cells for 24 h (b) and 48 h (c) The values of DPPH and ABTS were used as 1/IC₅₀ value. 4T1 cells were treated with 50, 100, 150, and 200 μg/ml ZEF, ZBF, and EGCG for 24 and 48 h. Cell viability was monitored by MTT assay. Data are expressed as mean±SD (n=3)

Fig. 4

ZEF, ZBF, and EGCG caused the accumulation of the 4T1 cells in the S and G₂/M phases 4T1 cells were treated with 50, 100, 150 and 200 μg/ml ZEF, ZBF, and EGCG for 24 h. The cells were washed, fixed, stained with propidiumiodide, and analyzed for DNA content by flow cytometry. (a) One representative result is shown. (b–d) Percentages of 4T1 cells in different phases of cell cycle after ZEF (b), ZBF (c), and EGCG (d) treatments. ^* P<0.05, ^** P<0.01, ^*** P<0.001 as compared with control (0 μg/ml)

Fig. 5

Effects of ZEF, ZBF, and EGCG on 4T1 cell migration by wound healing assay after 24 and 48 h incubation at 50, 100, 150, and 200 μg/ml (×100 magnification)

Fig. 6

Effects of ZEF, ZBF, and EGCG on 4T1 cell migration and invasion 4T1 cells were treated with 0, 50, 100, 150, and 200 μg/ml ZEF, ZBF and EGCG for 24 h. (a) Invasion images were taken with microscopy (×400 magnification). (b) Relative invasion ratio was calculated by counting three different images of migration cell numbers. ^* P<0.05, ^** P<0.01 as compared with control (0 μg/ml). Data are expressed as mean±SD (n=3)