Study On Differences In The Inhibitory Effects Of Different Phenolic Compound Fractions From Litchi Pulp On Hepatocyte Steatosis In Vitro And The Mechanism

Litchi(Litchi chinensis Sonn.) is a typical fruit in subtropical fruit with rich nutrition in the pulp, which has been as a traditional Chinese medicine to benefit the spleen, liver and skin. Modern researches have confirmed that phenolic-rich litchi pulp extracts exhibited significant antioxidant activity and improved lipid homeostasis in the high fat-fed mice. However, the extracts consisted of a variety of phenolic compounds so that the effective ones were not clear in those experiments. The present work were to compare the total phenolics, total flavoniods and tannin content and antioxidant activity of these fractions after separating litchi pulp polyphenol extracts into different phenolic compound fractions, then investigate the inhibitory effects of these fractions on lipids accumulation in vitro cell model of hepatic steatosis to identify the major bioactive phenolic compounds, and elucidate their molecular mechanism and the main signal pathway of improving hepatic steatosis by real time RT-PCR and western blotting. The main results in this study were presented as follows.1. Separation and antioxidant activity of different phenolic compound fractions from litchi pulpLitchi pulp polyphenol extracts were separated into four phenolic compound fractions(F1, F2, F3 and F4) by C18 silica gel column. Among them, the yield of F2 was as high as 36%, and others# were 18.71%, 16.79% and 21.12% respectively. Total phenolics, total flavonoids and tannin content of four phenolic compound fractions ranged from 218.86 to 499.78 mg GAE/g DW, 414.94 to 1285.45 mg RE/g DW and 83.35 to 483.43 mg CE/g DW respectively. Among four compound fractions tested, F2 exhibited the highest total phenolics, total flavonoids and tannin content with their corresponding percentage contribution as high as 50.3%, 54.2% and 72.1% to litchi pulp polyphenol extracts, followed by F3, F4, and finally F1. Procyanidin B2, epicatechin, A-type procyanidins trimer, B-type procyanidins dimers and trimer in F2, quercetin-3-O-rutinoside-7-O-a-L-rhamnoside in F3, and rutin, kaempferolrutinoside-rhamnoside, isorhamnetin-rutinoside-rhamnoside and isorhamnetin- rutinoside in F4 were identified by HPLC-DAD and LC-ESI-MS. The results of antioxidation indexes showed that FRAP values of four compound fractions were F2>F3>F4>F1(p<0.05). The IC50 values of DPPH free radical scavenging were 27.00, 9.76, 19.41 and 16.25 !g"m L-1 respectively, then F2 exhibited the strongest DPPH free radical scavenging ability with minimum IC50 value among four compound fractions, followed by F4, F3, and finally F1. Also, the ORAC values of them were F2>F3>F4>F1(p<0.05) and the CAA values were F2>F3>F4YF1(p<0.05). Among them, F2 exhibited the highest ORAC and CAA values as 8.36 mmol TE/g DW and 190.71 !mol QE/g DW with the percentage contribution as high as 50.4% and 84.9% to total ORAC and CAA values of litchi pulp polyphenol extracts respectively. These results indicated that there were significant differences in total phenolics, total flavonoids and tannin content and antioxidant activity between four phenolic compounds fractions from litchi pulp. The compositions of each phenolic compound fraction were also different. What#s more, F2 exhibited the highest yield, total phenolics, total flavonoids and tannin content with the best antioxidant capacities. Interestingly, the phenolics in F2 from litchi pulp may be the most main active compounds of antioxidant activity.2. Difference in the inhibitory effects of different phenolic compound fractions from litchi pulp on hepatocyte steatosis in vitroIn order to establish an vitro HepG2 cell model of oleic acid-induced hepatic steatosis, The triglyceride(TG) and total cholesterol(TC) content, levels of alanine aminotransferase(ALT) and aspartate aminotransferase(AST), malondialdehyde(MDA) content and superoxide dismutase(SOD) activity were measured in different concentration of oleic acid(OA) induced Hep G2 cells. The results have showed that OAK0.4 mmol/L exhibited a dose-dependent induction of TG and TC accumulation, and no cell damage was observed based on the changes in ALT and AST levels as well as MDA content and SOD activity. Therefore, 0.4 mmol/L OA was used to induce hepatocyte steatosis in vitro in the following experiments. The inhibitory effects of four phenolic compound fractions from litchi pulp on lipids accumulation in oleic acid-induced Hep G2 cells indicated that F2 exhibited the most efficient on suppressing TG accumulation with the percentage of suppression as high as 26.9% at 5 !g/mL, followed by F3 with the percentage of suppression as high as 26.2% at 10 !g/m L, next F4 with the maximal percentage of inhibition only as 19.3% at 20 !g/m L and finally F1 with the maximal percentage of inhibition only as 11% at 40 !g/m L from 5 to 40 !g/m L. The inhibitory effect of four fractions on TC accumulation was consistent with TG accumulation, namely F2>F3>F4>F1(p<0.05). In addition, F2 and F3 showed synergistic inhibition on lipids accumulation in HepG2 cell, while F4 show no synergy with F2 and F3. Implying, the compounds in F2 and F3 from litchi pulp may be the main active phenolics of improving hepatocyte steatosis.3. Molecular mechanism of the main phenolic compound from litchi pulp on hepatocyte steatosis in vitroThe phenolic compound fraction F3 from litchi pulp consisted of only a free phenol, quercetin-3-O-rutinoside-7-O-a-L-rhamnoside(QRR), identified as one of the main active phenolics of improving hepatocyte steatosis. The vitro cultured HepG2 cells were divided into control group, oleic acid model group and different concentration of QRR groups to elucidate the influence of the free phenol from litchi pulp on hepatic lipid metabolism-related genes and proteins in hepatocyte steatosis model by real time RT-PCR and western blotting. The results have showed that QRR could down-regulate in a dose-dependent manner the mRNA expression levels of hepatic scavenger receptor CD36 and fatty acid transport protein-2(FATP-2) as well as the protein expression level of FATP-2 to decrease the uptake of exogenous fatty acids into the liver cell(pS0.05). QRR significantly reduced the mRNA expression levels of diacylglycerol acyltransferase(DGAT1 and DGAT2), the key enzyme of triglyceride synthesis, as well as the protein expression level of DGAT1(p<0.05), but exhibited no significant influence on the protein expression level of DGAT2. Also, QRR could increase the phosphorylation of adenosine-5’-monophosphate(AMP)-activated protein kinase(AMPK), activate peroxisome proliferators activated receptor(PPAR) and up-regulated genes for fatty acid oxidation such as carnitine palmitoyl transferase-1(CTP-1), but not influence genes for fatty acid synthesis such as sterol regulatory element binding protein 1-c(SREBP1-c), Acetyl-CoA carboxylase(ACC) and fatty acid synthetase(FAS). In addition, QRR still repressed lipids accumulation in HepG2 cells through inhibiting AMPK activation by Compound C, which indicated that QRR could improve hepatocyte steatosis not entirely through AMPK. These results have showed that the mechanism for QRR in improving hepatocyte steatosis regulated the expression of genes and proteins mainly involved in fatty acid absorption, fatty acid oxidation and triglyceride synthesis. Importantly, AMPK/ PPAR pathway was one of the signal pathways in improving hepatocyte steatosis of QRR.