The Study On Antioxidant And Anti-obesity Of Pu-erh Tea

In this dissertation, the Pu-erh tea was successively extracted with acetone, water, chloroform, ethyl acetate, and n-butanol, the extracts were then isolated by column chromatography. The antioxidant effects of Pu-erh tea extracts and their isolated fractions were evaluated with different antioxidant experiment models. The author found some special polyphenols in Pu-erh tea, which had strong antioxidative activity, which was different from the catechins exits in green tea or the theaflavines in black tea. In the present study, the author focused on the Pu-erh tea special polyphenols' effects on the pre-adipocyte and adipocyte, as compared with the effect of epigallocatechin gallate(EGCG). The author obtained some valuable findings.1. Basic study on the Antioxidatnt activity of Pu-erh TeaBecause of their high reactivity, free radicals can damage diverse cellular macromolecules, including proteins, carbohydrates, lipids, and nucleic acids. Free-radical-caused damage to these molecules has been implicated in the causation of some degenerative diseases. The interest in the health properties of Pu-erh tea and related scientific investigation is increasing. Although a number of mechanisms have been proposed for the beneficial effects of Pu-erh tea in different models of chronic diseases, the radical scavenging and antioxidant properties of components in Pu-erh tea may be important contributors. In this study, the author successively extracted the Pu-erh tea with water, chloroform, ethyl acetate, n-butanol and 70% ethanol, consequently the Pu-erh tea was separated into five extracts; In order to investigate the main composition that responsive to the antioxidant effect of Pu-erh tea, the antioxidant effects were assayed by antioxidant experiments in vitro and animal modes in vivo.The main results were listed below:1) The strong scavenging effect to the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical and the hydroxyl radicals was observed in the ethyl acetate extract and the n-butanol extract.2) The antioxidant effects of green tea, black tea and Pu-erh tea on mice were all positive, while the effect of black tea and Pu-erh tea showed more powerful than the effect of green tea. Furthermore, the positive antioxidant effect was observed in the fractions extracted with ethyl acetate and n-butanol of Pu-erh tea, and the fraction precipitated with 70% ethanol, ie, tea polysaccharide, but in the residual water soluble fraction, non antioxidant effect was found in mice.These results suggested that the ethyl acetate extract and the n-butanol extract which contributed to the scavenging effect on free radicals in vitro, were the main antioxidant components in Pu-erh tea. The ethyl acetate extract, the n-butanol extract and the fraction precipitated with 70% ethanol were the main composition that responsive to the antioxidant effect of Pu-erh tea in vivo.2. The study on the special polyphenols in Pu-erh teaThe main difference of Pu-erh tea from green tea and black tea is the special processing of Pu-erh tea, and there maybe some special polypehnols generated in the processing. In the present study, the author extracted the Pu-erh tea with acetone, and got the acetone soluble fraction (ASF), then successively re-extracted ASF with chloroform, ethyl acetate and n-butanol. The extracts were isolated by modern chromatogram, according to the thin layer chromatography (TLC) results. The Pu-erh tea ethyl acetate extract was then fractionated into eight fractions, assigned as PEF1 to 8, similiary the n-butanol extract was fractionated into five fractions, assigned as PBF1 to 5. The author tested the antioxidative activities of these fractions for their inhibitory effect on Fenton reaction system and superoxide anion-generating system; assessed the protective effect and reactive oxygen species (ROS) inhibiting effect of Pu-erh tea extract's fractions on hydrogen peroxide-induced oxidative damage in the HPF-1 cells; In order to comprehensively investigate the antioxidant effect of special polyphenols in Pu-erh tea, the author assayed the scavenging effect on singlet oxygen, DPPH, 2,2 -Azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS⁺), assayed the inhibition effect on lipid per-oxidation, and compared with the effect of EGCG.The main results were listed below:1) Pu-erh tea ethyl acetate extract, n-butanol extract and their fractions showed scavenging activity on OH^·and O₂^·, the fractions PEF2, PEF8, PBF3, PBF4 and PBF5 showed protective effect against H₂O₂, and against high concentration glucose-induced damage in HPF-1 cells. These fractions increased the cells viability under normal conditions. In addition, it was found that these fractions, except PBF5, decreased the accumulation of intracellular ROS in H₂O₂-induced cells.2) The antioxidant effect of PEF8 on the above four systems is much stronger than that of EGCG, a typical green tea catechin, but there is almost no monomeric polyphenols, theaflavins and gallic acid in the PEF8 when assayed by HPLC and other methods.3) The scavenging effect on singlet oxygen, DPPH, ABTS⁺, and the inhibition effect on lipid per-oxidation of PEF8, was approximately close to the effect of EGCG.From these results, the author believed that Pu-erh tea has strong antioxidant activity. There maybe some special polyphenols generated in the special processing of Pu-erh tea, and their strong antioxidant ability may be important contributor of the beneficial effects of Pu-erh tea.This research will be useful for us to know more about the beneficial effects of Pu-erh tea and their special components. Moreover, the beneficial oxidation of the tea polyphenols induced by humidification and caloric in the process of piling and long time preservation of Pu-erh tea, maybe an interested introduction for us to investigate the beneficial oxidation of the other antioxidants.3. Pu-erh tea extracts modulated the physiological function of 3T3-L1 pre-adipocyte and mature adipocyteGreen tea polyphenols, including EGCG, attenuated obesity and related diseases by various mechanisms, one pathway may be the direct modulation on the physiological function of 3T3-L1 pre-adipocyte and mature adipocyte. Many animal experiments and cell experiments showed that Pu-erh tea has the anti-obesity effect, and the author found that the PEF8 in Pu-erh tea has strong antioxidant activity in the above experiments. In this study, the author investigated the modulation of PEF8 on the physiological function of 3T3-L1 pre-adipocyte and mature adipocyte, and compared with the effect of EGCG.In the present study, the modulation of PEF8 and EGCG on the pre-adipocyte cell viability, apoptotic and proliferation were investigated; The ROS level, the expression of apoptotic relative proteins, the expression of adipogenesis relative proteins, the content of glycerol and triglyceride in the cells were investigated after the treatment with PEF8 or EGCG.The main results were listed below:1) PEF8 and EGCG inhibited the 3T3-L1 pre-adipocytes viability in time- and concentration-dependent manner. PEF8 also inhibited the 3T3-L1 pre-adipocytes proliferation in the same manner. When the pre-adipocytes were treated with the PEF8 or EGCG in pre-confluent (logarithmic growth phase, about 70%) for 48h, a great deal of apoptosis were initiated, but only a little were initiated when the pre-adipocytes were treated after the confluent.2) EGCG (2μg/ml to 50μg/ml) reduced the ROS level when the pre-adipocytes were treated in pre-confluent for 3h, but as the concentration of EGCG increased, the loss of ROS decreased inversely. PEF8 (2μg/ml to 10μg/ml) can also reduced the ROS level when the pre-adipocytes were treated in pre-confluent, but when the treated concentration of PEF8 were 20μg/ml and 50μg/ml, the ROS level were higher than that of the untreated pre-adipocytes.3) When the pre-adipocytes were treated in pre-confluent with PEF8 or EGCG for 48h, the expression of TNFαand NF-κB were up-regulated, the expression of P-JNK, C/EBPβand C/EBPαwere down-regulated.4) When the pre-adipocytes were treated in post-confluent with PEF8 or EGCG for 48h, the expression of PPARγwas up-regulated, and the adipogenesis increased in concentration-dependent manner.5) PEF8 and EGCG promoted lipid metabolism in the mature adipocytes which were treated for 48h, the number and the type of adipocytes, the content of triglyceride in the adipocytes decreased consequently.These results showed that the decrease of cell viability and proliferation, the apoptotic of pre-adipocytes when treated with PEF8 or EGCG, were induced by the improvement of ROS level, the up-regulated expression of TNFαand NF-κB and the down-regulated expression of P-JNK, simultaneously, through such treatment, the adipogenesis relative proteins were also down-regulated consequently. The sensitivity of pre-adipocytes to the treatment of PEF8 or EGCG were different between different growth phases (pre-confluent and post-confluent), PEF8 or EGCG could up-regulated the expression of PPARγwhen the pre-adipocytes were treated in post-confluent subsequently, the pre-adipocytes differentiation promoted, and the content of triglyceride also increased. At the same time, however, PEF8 or EGCG can also promote lipid metabolism in the mature adipocytes, decrease the number and the type of adipocytes, and decrease the content of triglyceride in the adipocytes.