| PGG(1,2,3,4,6-Penta-O-Galloyl-Beta-D-glucose) is widely exsited in fruits,vegetables and herbal plants, and has a variety of bioactivities. It also has the functions of immunoregulation and health care of human body.Our team has extracted PGG from eucalyptus leaves and proved the anti-oxidation,anti-tumor and anti-aging effects of which, but the digestibility, metabolic pathway and the degradation products are still remain invalid, and require further study. This paper used the human intestinal flora to vitro anaerobic fermentation of PGG.Degradation products and terminal products of PGG were separated and purified, also the structures and degradation pathways of these products were detected.Some important degradation products of PGG were separated and purified by preparativechromatography and their antioxidant capacity were evaluated.The purpose of this paper is to provide a basement for exploring the bioloactivity mechanism of PGG. The research and results of this paper are as follows:(1)Antioxidant activity of PGG.DPPH?ABTS radical scavenging experiments?and ORAC experiments were used to evaluate the antioxidative ability. The results showed that PGG had good antioxidant ability, which could effectively inhibit the free radical of DPPH and ABTS in the solution. And PGG exhibited significantly higher activities than the Trolox and Vc,indicating that PGG has a strong antioxidant activity.(2)Evaluation of in vitro antioxidant chemical although non-cellular. The oxidative damage model of PC12 cells was established in this paper. The results showed that: PGG had no toxie effect on cultured PC12 cells under the concentrations of 25~100?g/m L. Cells treated by PGG increased the survival rate of cells, therefore, PGG has a significant protective effect on cell injury.(3)Anti tumor activity of PGG.Selected HCT-116 cells as the research object, after treatment with PGG,cell proliferation was determined by MTT assay. Cell cycle, cell apoptosis were determined by flow cytometry.It was found that the proliferation of colon V cancer cells treated with different concentrations of PGG was inhibited, and PGG inhibited cell proliferation in a dose and dependent manner. In the flow cytometry analysis, PGG treatment in 0~20?mol/L caused the number of colon cancer S cells increased, indicating that PGG can cause significant S phase arrest. We also can see that the number of early apoptosis increased significantly, so the PGG can effectively induce apoptosis of colon cancer cells.(4)Study on preparation of PGG and the stability of which in the simulated gastrointestinal p H environment.The hydrolysable mixture of PGG was extracted from eucalyptus leaves by methods such as solvent extraction, macroporous adsorption resin, medium pressure reversed-phase silica gel and preparation liquid phase.The purity of which was detected by HPLC.PGG was added into stimulated gastric(PH=1 buffer solution) and intestinal fluid(PH=7.2 buffer solution) to determine the stability of which in the gastrointestinal tract environment.The result of HPLC showed that the purity of separated and purified PGG>95%,and could be used in subsequent tests.In 0-6h,PGG had good stability in simulated gastric fluid p H buffer solution while slightly degradated in simulated intestinal fiuid p H buffer solution, but also stabilized after 2h.The result showed that PGG had good stability in acidic environment and weak alkaline environment.(5)Study on anaerobic degradation of PGG in intestinal flora.Culture solution was prepared to vitro anaerobic culture of intestinal flora.PGG with specific concentration was added in culture solution with intestinal flora, and anaerobically cultured at 37 ? in certain time.Then take the fermented liquid from different time periods to centrifugal separation,the supernatant was extracted by ethyl acetate. Dried extracts were detected by HPLC.The results obtained were as follows:7 compounds were named according to the degradation order as P1, P2, P3, P4, P5, P6, P7.P7 was the terminal product of degradation of PGG. With the concentration of PGG increased, the concentration of P7 also increased.The amount of P1-P6(degradation intermediates) and P7(terminal products) were changed with time between 1-24 h.(6)Verification of the results of PGG's tannin enzyme hydrolysis.In order to verify the degradation of PGG in the intestinal flora may be related to the enzyme of intestinal flora,the product of PGG digested by PGG tannin enzyme was extracted by ethyl acetate.Then the dried extract was detected by HPLC.The HPLC spectrums of extract were compared with the HPLC spectrums of PGG intestinal flora fermentation liquid.The results showed that the peak order and peak time was the same in the degradation products and PGG hydrolized by enzymes.The terminal product of the enzyme-hydrolyzed PGG was P6, and the degradation of PGG may be related to the tannin enzyme of the intestinal flora.Since P6 was the intermediate product while P7 was the terminal product,so the degradation effect was not only took by a single tannin enzyme in fermentation liquid, but also other enzymes contributed to it.(7)Isolation, purification and structure identification of PGG degradation products.Seven PGG degradation products were isolated and enriched by preparative chromatography.The molecular weight of PGG degradation products was detected by ESI-MS.The molecular weight was used to predict molecular structure.The results showed that the molecular weight of PGG degradation products were 788,636,484,332,170 and 126 respectively.The molecular weight of P2 and P3 were both 636 which means they could be isomers.The molecular weight of P1,P2(P3),P4 and P5 were having a difference of one gallic acid residue(152) by turn.It is speculated that these compounds may be followed by TGG(4GG), Tri GG(3GG), DGG(2GG) and MGG(1GG).P6 was gallic acid(GA) and P7 was pyrogallol(PGA).The structure of P1 to P5 remains to be further determined.(8)Detection of antioxidant activity of PGG degradation products.The antioxidant activity of PGG degradation products was evaluated by methods such as ABTS,ORAC and DPPH.The results were as follows: In DPPH free radical scavenging test,the concentration of each compound required was Tri GG> DGG> PGG> TGG> GA> MGG> PGA when scavenging rate was 50%.Tri GG and DGG IC50 values did not have significant differences in this test.In ABTS free radical scavenging test,all degradation products' IC50 values were less than PGG's.In ORAC test,the antioxidant capacity was GA>PGG>PGA>MGG, while TGG, Tri GG, DGG and PGG were not significantly different from each other.PGG's degradation products had strong antioxidant capacity was proved by three antioxidant activity evaluation test.It showed that PGG did not decrease the antioxidant activity after fermentation and degradation by intestinal flora. |
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