The enzymatic synthesis of theaflavin-3-gallate oxidation product and its determination.

In this work, the oxidation of theaflavin-3-gallate (TF-3-G) was investigated using (-)-epicatechin (EC) and (-)-epigallocatechin gallate (EGCG) as substrates in a one-pot reaction. The resulting TF-3-G oxidation product was acquired by employing acetonitrile/water and ethanol/water as eluents, respectively, which was identified as theanaphthoquinone-3'-gallate (TNQ-3'-G). Surprisingly, we discovered that TNQ-3'-G could react with certain protic solvents to form new and unstable complexes through intermolecular hydrogen bond. This reactivity was also confirmed by the presence of irregular peaks in reverse-phase high-performance liquid chromatography (RP-HPLC) besides spectroscopic data. Therefore, we inferred that the number of carboxyl groups may increase through the successive oxidative polymerization of the TFs oxidation products. The high-molecular polymer could also interact with biomacromolecules in a similar manner to their interaction with protic solvents. This interaction might be one of the main factors contributing to the broad hump of thearubigins (TRs) on the RP-HPLC baseline. Additionally, these findings lay a solid foundation for interpreting the structures of TRs and understanding their generation mechanism.

Efficient enzymatic synthesis of theaflavin and its production mechanism.

In this study, a novel preparation method of theaflavin (TF) has been established. Our findings indicated that the formation of TF was significantly enhanced by using an ice bath (2-3°C). Additionally, increasing the ratio of (-)-epigallocatechin (EGC) under the ice bath could further improve its yield. This approach prevented the appearance of a dark solution within 3 h, effectively protecting TF from oxidation. Our study on the generation mechanism of TF suggested that EGC-quinone I (EGC-Q-I) with two carbanions could potentially serve as one of synthons based on the retrosynthetic analysis of the bicyclo[3.2.1]octane-type intermediate. Subsequently, quantum mechanical calculations further supported this hypothesis. Practical Application: In this study, we have developed a novel method for the synthesis of theaflavin (TF), demonstrating that the use of ice bath significantly enhanced its yield. Increasing the ratio of (-)-epigallocatechin (EGC) under the ice bath further improved TF yields and prevented darkening of the solution for at least 3 h, thereby protecting TF from oxidation. Our study suggested that EGC-quinone I is a potential synthon based on the retrosynthetic analysis of the bicyclo[3.2.1]octane-type intermediate (BOI). This hypothesis is supported by QM calculations.

The Effect of Different Treatments of (-)-Epigallocatechin-3-Gallate on Colorectal Carcinoma Cell Lines.

Backgroud: (-)-Epigallocatechin-3-gallate (EGCG), the major component of green tea, is well documented to induce apoptosis and cell cycle arrest in cancer by targeting multiple signal transduction pathways. However, EGCG is extremely unstable in general culture conditions and rapidly degraded. So, to what extent EGCG or which degradation products of EGCG play a role in anti-tumor is still unknown. In this study, we evaluated the effect of different treatments of EGCG on HCT116 cells. DESIGN: MTT assay was applied to evaluated the inhibitory effect of different treatments of EGCG on HCT116 cells. Cell cycle and apoptosis were performed by flow cytometry. Finally, western blot analysis was used to elucidate the molecular mechanism associated with cell cycle arrest and apoptosis. RESULTS: Compared with control, both EGCG and O-EGCG (i.e., EGCG being pre-incubated at 37°C for 3 h) significantly inhibited HCT116 cells growth. Surprisingly, we found that the inhibitory effect of O-EGCG was stronger than that of EGCG. The IC50 values of EGCG and O-EGCG were 8.75 and 5.40 µM, respectively. Cell cycle analysis showed that 20 µM of EGCG simultaneously caused cell cycle arrest at G1 and G2 phase in HCT116 cells, differing from O-EGCG which exclusively caused cell cycle arrest at G2. This result suggested that parent EGCG at the early treatment might cause cell cycle arrest at G1. As time went on, EGCG disappeared and degraded products of EGCG were formed which might cause cell cycle arrest at G2. Further studies revealed that EGCG induced cell cycle arrest at G1 by downregulation of cyclin E and cyclin D1 and upregulation of p21. On the other hand, O-EGCG induced HCT116 cells apoptosis mainly by increasing the expression of p53 and cleaved caspase-3, which might be the underlying reason why O-EGCG had stronger inhibitory effect on HCT116 cells line than EGCG. CONCLUSIONS: The pretreatment of EGCG may be an effective way to enhance its antitumor effect.

Lactobacillus paracasei ssp. paracasei YBJ01 reduced d-galactose-induced oxidation in male Kuming mice.

We investigated in vitro and in vivo antioxidant activity of Lactobacillus paracasei ssp. paracasei YBJ01 (LPSP-YBJ01) isolated and identified from fermented yogurt. Strain LPSP-YBJ01 had stress tolerance against acidity, bile salt, and osmotic pressure. Five in vitro antioxidant assays were used to evaluate antioxidant activity of LPSP-YBJ01, which could scavenge free radicals (2,2-diphenyl-1-picrylhydrazyl and hydroxyl) and superoxide anion in vitro. In addition, strain LPSP-YBJ01 had stronger antilipid peroxidation activity and weak reducing power in vitro. We measured in vivo antioxidant activity of LPSP-YBJ01 in an oxidation mouse model induced by d-galactose injection. Strain LPSP-YBJ01 significantly increased serum superoxide dismutase (SOD), glutathione peroxidase, and total-antioxidant capability, and inhibited generation of malondialdehyde in a dose-dependent manner. In addition, strain LPSP-YBJ01 also increased the hepatic and splenic protein expressions of some antioxidant enzymes such as catalase, Cu/Zn-SOD, and Mn-SOD in mice treated with d-galactose. Thus, LPSP-YBJ01 had antioxidant activity in vitro and in vivo and may be a useful probiotic.

The influence of pH and enzyme cross-linking on protein delivery properties of WPI-beet pectin complexes.

The incorporation of bioactive proteins and peptides into food is associated with the loss of bioactivity due to deactivation in complex food matrices and in digestion systems. In this study, two different types of protein carriers, i.e. biopolymer complexation and complex coacervation were fabricated using whey protein isolation (WPI, 6wt%) and beet pectin (BP, 1.25 and 1.00wt%) at pH5.5 and 3.5, respectively. The release of the encapsulated FITC-BSA, a model bioactive protein, in both carriers in the absence and presence of laccase was investigated at both pH7.0 and 4.0. Release of FITC-BSA from both lyophilized WPI-beet pectin biopolymer complexation and complex coacervation were biphasic with an initial burst release followed by a slower release phase. The addition of laccase in biopolymer complexation increased the loading efficiency from 44.95% to 52.15% and slowed down the burst release of FITC-BSA but did change the biphasic release pattern. Laccase-cross linked WPI (6wt%)-BP (1wt%) complex coacervation had highest FITC-BSA loading efficiency (96.90%). The release of the embedded FITC-BSA in this carrier at both pH4 and 7 was in a gradual manner and the profile can be fit to zero order kinetics over the 72h study period suggesting enzymatically reinforced complex coacervation between the protein and the negatively charged beet pectin can restrain the burst release of FITC-BSA. These results indicate that laccase cross-linked WPI-beet pectin complex coacervation can be a good carrier system for delivering hydrophilic bioactive proteins or peptides successfully with enhanced loading parameters and sustained release profiles.

Pre-treated theaflavin-3,3'-digallate has a higher inhibitory effect on the HCT116 cell line.

The pro-apoptotic and inhibitory effects of the aflavin-3,3'-digallate (TFDG), which is the typical pigment in black tea, have been demonstrated in many cancer cell lines. However, TFDG is not stable in general culture conditions. So, to what extent TFDG or which degradation products of TFDG play an antitumor role is still unclear. In this study, we evaluated the effect of different treatments of TFDG on HCT116 cells. Compared with the control, both TFDG and O-TFDG (the TFDG that was pre-incubated in an incubator at 37°C for 3 hbefore adding into 96-well plates) significantly inhibited HCT116 cell growth. However, pre-treated TFDG was far better than TFDG. The IC50 values of TFDG and O-TFDG-3 were 17.26 µM and 8.98 µM, respectively (the cells were treated by O-TFDG for only 3 h, after which the media were replaced by fresh media for another 69 h incubation). Cell-cycle analysis revealed that 20 µM of O-TFDG and O-TFDG-3 caused cell-cycle arrest at G2 phase in HCT116 cells. Western blot analysis also demonstrated that the anti-inflammatory effect of O-TFDG-3 is stronger than that of TFDG by decreasing COX-2 and iNOS. On the other hand, O-TFDG induced HCT116 cells apoptosis mainly by increasing the expression of p53, p21, and cleaved caspase-3. The current study demonstrated that O-TFDG had a higher inhibitory effect on HCT116 cells than TFDG, and sowe may inferfromthis that the degradation products of TFDG play a key role against tumors.

[Progress in catechins oxidation products and their formation mechanism].

Catechins are the key components of tea and have a great impact on its quality. Catechins can be oxidized to form a new black tea polyphenols, some of which have better pharmacological effect. However, the formation mechanism of these new polyphenols is still unclear. In this paper, oxidation products coming from catechins and the formation mechanism of the new compounds are reviewed.It is the base of further study on theaflavins, thearubigins and theabrownines.

Lipid oxidation in base algae oil and water-in-algae oil emulsion: Impact of natural antioxidants and emulsifiers.

The impact of natural hydrophilic antioxidants, metal chelators, and hydrophilic antioxidant/metal chelator mixture on the oxidative stability of base algae oil and water-in-algae oil emulsion was investigated. The results showed that green tea extract and ascorbic acid had greatest protective effect against algae oil oxidation and generated four day lag phase, whereas rosmarinic acid, grape seed extract, grape seed extract polymer, deferoxamine (DFO), and ethylenediaminetetraacetic acid (EDTA) had no significant protective effect. Besides, there was no synergistic effect observed between natural antioxidants and ascorbic acid. The emulsifiers are critical to the physicochemical stability of water-in-algae oil emulsions. Polyglycerol polyricinoleate (PGPR) promoted the oxidation of emulsion. Conversely, the protective effect on algae oil oxidation was appreciated when defatted soybean lecithin (PC 75) or defatted lyso-lecithin (Lyso-PC) was added. The role of hydrophilic antioxidants in emulsion was similar to that in algae oil except EDTA which demonstrated strong antioxidative effect in emulsion. The results could provide information to build up stable food products containing polyunsaturated fatty acids (PUFA).

[Advance in anticancer studies on catechins and their derivatives].

Experimental studies on animals discovered that catechins have a notable inhibitory effect on the formation and development of tumors in different organs. Studies on their mechanism showed that catechins could inhibit cancer cell invasion and proliferation, angiogenesis and metastasis and induce cancer cell apoptosis through anti-oxidation and by inhibiting related enzyme activities and cancer cell signal transmission. This essay provides an overview of correlation between tea drinking and cancer prevention, various mechanisms of catechins and their derivatives such as biochemical cancer prevention and anticancer and looks into the future of catechins.