Studies On The Mechanism Of Antioxidant And Antitumour Activities Of Functional Compositions From Selenium-Enriched Green Tea

As green tea possessed the antioxidant and antitumor activities and the plant-source Sehad the potential of chemo-antitumor activity, green tea was screened as the Se enrichingcarrier and different components of tea polyphenols, tea polysaccharides and tea proteinswere prepared from both Se-enriched and regular green tea using the advanced separationtechnology of bioactive substances. Then, their antioxidant activities were compared toprimarily screen the high bioactive components. Furthermore, incubation model of humanhepatocarcinoma cell lines HepG2 was established by advanced biological technology andmeans to study in vitro antitumor activity of the selected component. The main researchresults were as follows:1. Analysis physiochemical properties of Se-enriched green tea indicated that Se couldgreatly improve the tea sensory quality, enhance the tea color, decrease the tea bitternessand increase the proteins and free amino acids contents. However, no differences werefound in tea polyphenols and chlorophyll contents. According to different extractionprocedures, crude tea polyphenols, crude tea polysaccharides and crude tea proteins wereprepared and their purities were 84.11%, 40.30% and 62.49%, respectively. There was nosignificant difference in each component except the protein content (P＞0.05).2. Two different systems to assess the antioxidant power of different components andwater extracts of the regular green tea and Se-enriched green tea, AAPH and DPPH methodwere applied. Both systems indicated that extracts of Se-enriched green tea possessed asignificant higher antioxidant activity than that of regular green tea. The ability to inhibitlipid peroxidation was decreased in the following order: crude tea proteins＞crude teapolyphenols＞crude tea polysaccharides by AAPH method. However, their power toscavenge DPPH radical followed the order: crude tea polyphenols＞crude tea proteins＞ crude tea polysaccharides by DPPH method. Furthermore, their EC₅₀ values were measuredand it was 22.43±0.13μg mL^-1 for tea extracts, 4.03±0.01μg mL^-1 for crude teapolyphenols, 273.33±8.66μg mL^-1 for crude tea polysaccharides, 38.36±2.94μg mL^-1 forcrude tea proteins of Se-enriched green tea and 16.65±5.34μg mL^-1 for a-tocopherol. Thecrude tea polyphenols and crude tea polysaccharides of Se-enriched green tea possessed asignificant higher antioxidant activity than that of regular green tea. But there was nodifference for tea proteins (P＞0.05).3. Polyphenols from Se-enriched green tea were purified by silica-gel columnchromatography and the main catechin monomer ingredient EGCG content was 50.93%with Se content of 0.29±0.012 mg kg^-1, significantly higher than that of regular green tea,respectively (46.90% and 0.052±0.018 mg kg^-1). EGCG content of Se-enriched green teawater extracts was 12.39% with Se content of 4.030±0.005 mg kg^-1, significantly higherthan that of regular green tea, respectively (11.65% and 0.037±0.002 mg kg^-1). Polyphenolsand water extracts from Se-enriched green tea exhibited significantly higher antioxidantactivity than the same components from regular green tea plus sodium selenite.4. Tea polyphenols and water extracts from Se-enriched and regular green tea andpolyphenols and water extracts from regular green tea plus selenite possessed greatdose-dependent antitumor effect on A549 cells in the range between 50μg mL^-1 and 200μgmL^-1 and on HepG2 cells ranged from 200μg mL^-1 to 1000μg mL^-1. At the same Secontent, tea polyphenols and water extracts from Se-enriched green tea possessedsignificantly higher antitumor activity than polyphenols from regular green tea and waterextracts plus selenite(P＜0.05). The IC₅₀ values of tea polyphenols and water extracts fromSe-enriched green tea were 109.16±0.17μg mL^-1 and 217.88±0.26μg mL^-1, respectivelyand tea polyphenols from Se-enriched green tea exhibited greater antitumor activity thanwater extracts.5. Visible and fluorescence staining results showed that HepG2 cell performed thetypical apoptosis character of cell lessening, cytomembrane collapse, karyopyknosis,inregular changes of caryotype and nuclear fragmentation after addition of polyphenolsfrom Se-enriched green tea, which indicated polyphenols from Se-enriched green teacould undoubtedly induce the apoptosis of human hepatocarcinoma cell lines HepG2. Under the same dosage, tea polyphenols from Se-enriched green tea induced strongerdamage to HepG2 cell than that of water extracts.6. The effect of tea polyphenols from Se-enriched green tea on cell cycle of HepG2was measured with flow cytometry. After the treatment for 24 h at 250μg mL^-1, theapoptotic cells at the early and late stage accounted for 14% and 1.6% of the totalcounted cells, significantly higher than the control of 1.8% and 0.2%, respectively. Teapolyphenols from Se-enriched green tea could keep the cell cycle of HepG2 cells to S stage(control 24.59%, treatment 52.49%), block cell cycle to G2-M, inhibit the proliferation andinduce the apoptosis of the cells.Effects of tea polyphenols from Se-enriched green tea on protein expression of Bcl-2,Bax were investigated by western blots. At 500μg mL^-1, it could decrease the expression ofapoptosis inhibitor gene Bcl-2 and promote the expression of apoptosis induction gene Bax.The ratio of Bax/Bcl-2 was 1.14, significantly higher than the control of 0.75. Waterextracts of Se-enriched green tea couldn't greatly decrease the expression of Bcl-2.However, at 5μg mL^-1, it could promote the expression of Bax. And the ratio of Bax/Bcl-2 was 1.49, significantly than the control of 1.07.