| Acute liver injury is a very common liver disease. The main treatment measures include administration of antioxidants and suitable support treatment. Acute liver damage is primarily induced by one of several causes, among them free radicals oxidative damage, viral exposure, alcohol consumption, and drug and immune system issues. Many studies show that the high contents of reactive oxygen species(ROS) and free radicals in the human body will cause liver cell membranes damage and DNA damage, and then cause acute liver injury. Therefore, it has important scientific significance and application prospects to screen extensively the safe and highly effective antioxidants, to study the biological effects and mechanisms, and to guide the use of the clinical drugs.Phloretin, a natural dihydrogen chalcone flavonoid found in the skin of apples, pears and a variety of vegetable juice, has good anticancer, antioxidant and antityrosinase activities. Phloretin can fade melanin, and consequently induce skin whitening better than kojic acid and arbutin. Phloretin is commonly used as a new type of whitening agent in cosmetics. In addition, phloretin has several biological activities such as anticancer, fall blood sugar, protect blood vessels and other activities. However, phloretin will be easily oxidated under the natural state, which greatly limits its application. It is a very significant work to modify the molecule structure of phloretin, to increase its stability, to save or improve antioxidant and antityrosinase activities. Phloretin isonicotinyl hydrazone(PIH) is a new compound synthesized by our team based on this idea. Now there are many studies about antioxidant and antityrosinase activities of phloretin, but there are rare studies about acute liver injury and anticancer mechanism treated with phloretin and PIH. Based on this idea, this thesis carried out the related research from this respect.Based on the prophase work of our laboratory, quercetin used as the reference, this paper studied the antioxidant and inhibit tyrosinase activities of phloretin and PIH, which is a new compound synthesized by our team. This paper also examined the hepatoprotective effects of phloretin and PIH on D- galactosamine or thioacetamide or acetaminophen-induced acute liver damage in Kunming mice as well as the possible mechanisms.This study also investigated the anticancer mechanism in BEL-7402 cells treated with phloretin and PIH using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay, flow cytometric(FCM)analysis, gelelectrophoresis analysis, western blot analysis, and real-time polymerase chain reaction(PCR).The results show that phloretin and PIH have good antioxidant activities. The antioxidant activity order is PIH>phloretin, in terms of the antioxidant models. The antityrosinase activity order is phloretin > PIH, in terms of the antityrosinase models. The hepatoprotective activity order is PIH>quercetin>phloretin, in terms of the liver injury models, while quercetin is used as the control drug. In terms of the experiment of the apoptosis mechanism of human liver cancer BEL- 7402 cells, the results show that phloretin and PIH induce cytotoxicity and apoptosis in BEL-7402 cells. Both intrinsic and extrinsic apoptosis pathways in addition to p53 signaling pathways, Bcl-2 family mitochondrial signaling pathways, and the stability of telomeres, controlled by h TERT, are involved in BEL-7402 cells apoptosis.Above all, phloretin and PIH have good antioxidant and antityrosinase activities, have the ability to protect the acute liver injury. Phloretin and PIH can also induce cytotoxicity and apoptosis in BEL-7402 cells. These results support the use of phloretin and PIH in acute liver damage and cancer chemoprevention and chemotherapy as the new drug candidates.Chapter 1 The introduction firstly introduces the current research status of phloretin, acyl hydrazone compounds, and tyrosinase. Then this paper introduces the principle of acute liver injury, the common drugs for animal model, and the research progress of natural medicine against acute iver damage. This paper also introduces the apoptosis mechanism of liver cancer, mainly including the various signal transduction pathways in the present study, and the research progress of apoptosis induced by traditional Chinese medicine(TCM) and flavonoids.Chapter 2 The purpose of this study is to examine the antioxidant activity of phloretin isonicotinyl hydrazone, quercetin, hloretin, silybin. The experiment examined the antioxidant effects of phloretin, quercetin, and PIH on lipid peroxidation in rat liver mitochondria in vitro, 1,1-diphenyl-2-picrylhydrazyl(DPPH) or,2-azino-bis-(3- thylbenzthiazoline-6-sulphonic acid)(ABTS) free radical scavenging activity in vitro, and supercoiled p BR322 plasmid DNA were confirmed. The experiment results showed that the four flavonoids had excellent antioxidant activity with the suitable concentration. In the experiment of scavening DPPH, ABTS radicals and inhibiting lipid peroxidation, the IC50 of phloretin isonicotinyl hydrazone was respectively 1.92 μmol/L,2.27μmol/L, 2.08μmol/L. The IC50 of quercetin was respectively 3.07μmol/L, 3.64μmol/L, 6.67μmol/L. The IC50 of phloretin was respectively 7.69μmol/L, 4.54μmol/L, 12.50μmol/L. The IC50 of silybin was respectively 96.15μmol/L, 7.10μmol/L,104.16μmol/L. These compounds provided dose-dependent protection against AAPH-induced free radical damage. The inhibition effects produced by these compounds(10 m M) are shown in the order of activity: PIH > quercetin > phloretin. The results show that phloretin and PIH have good antioxidant activities. The antioxidant activity order is PIH > quercetin > phloretin, in terms of the antioxidant models.Chapter 3 The purpose of this study is to examine the antityrosinase activity of phloretin and phloretin isonicotinyl hydrazone. The experiment examined the IC50 values, inhibitory type, and the values of KI and KIS of phloretin, and PIH on mushroom tyrosinase for the oxidation of L-DOPA. The experiment results showed that the IC50 values of quercetin, phloretin, and PIH on diphenolase activity were 25μmol/L,37.5μmol/L, and 50μmol/L, respectively. The activity order was quercetin > phloretin > PIH. Phloretin and PIH were reversible inhibitors of tyrosinase for the oxidation of L-DOPA. Phloretin and PIH were competitive-uncompetitive mixed type inhibitors. The values of KI and KIS of phloretin were determined as 23.5μmol/L and 129μmol/L, respectively. The values of KI and KIS of PIH were determined as 57.5μmol/L and 187.1μmol/L. The experiment results showed that phloretin and PIH had obvious inhibitory effects on the diphenolase activity of mushroom tyrosinase. Phloretin and PIH were competitive-uncompetitive mixed type inhibitors. The antityrosinase activity order is phloretin>PIH, in terms of the antityrosinase models.Chapter 4 The purpose of this study is to examine the hepatoprotective effects of phloretin, quercetin, and phloretin isonicotinyl hydrazone. This investigation examined the hepatoprotective effects of phloretin and phloretin isonicotinyl hydrazone(PIH) on D-galactosamine(D-Gal N) or thioacetamide(TAA) or Acetaminophen( AP)-induced acute liver damage in Kunming mice, as well as the possible mechanisms. The serum levels of alanine aminotransferase(ALT), aspartate aminotransferase(AST), γ-glutamyl transferase(γ-GT), alkaline phosphatase(ALP), and total bilirubin(TB) as well as the histopathological changes in mouse liver sections were determined. Phloretin, quercetin, or PIH significantly prevented the increase in serum ALT, AST, γ-GT, ALP, and TB in acute liver damage induced by D-Gal N, TAA or AP, and produced a marked reduction in the histopathological hepatic lesions. The experiment results showed that phloretin, quercetin, or PIH had obvious hepatoprotective effects on acute liver damage. The hepatoprotective activity order is PIH>quercetin>phloretin, in terms of the liver injury models, while quercetin is used as the control drug.Chapter 5 The purpose of this study is to test the cytotoxic effects of phloretin and PIH on BEL-7402 cells and to identify the underlying molecular pathways. This study investigates the anticancer mechanism in BEL-7402 cells treated with phloretin and PIH using the 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide(MTT) assay, flow cytometric(FCM)analysis, gel electrophoresis analysis, western blot analysis, and real-time polymerase chain reaction(PCR). The anticancer mechanisms were as follows: inhibition of cell proliferation, blocking of cell proliferation in the G0/G1 phase of the cell cycle, degradation of the genomic DNA, reduction of the amount of human telomerase reverse transcriptase(h TERT), p53(mutational), and Bcl-2 proteins, increase in the amount of Bax protein, and inhibition of the h TERT m RNA expression in BEL-7402 cells. Therefore, this study first investigates the anticancer activity and the expressions of h TERT, p53, Bcl-2, and Bax proteins in BEL-7402 cells treated with PIH. The results show that phloretin and PIH induced cytotoxicity and apoptosis in BEL-7402 cells. Both intrinsic and extrinsic apoptosis pathways in addition to p53 signaling pathways, Bcl-2 family mitochondrial signaling pathways, and the stability of telomeres, controlled by h TERT, are involved in BEL-7402 cells apoptosis. These results support the use of phloretin and PIH in cancer chemoprevention and chemotherapy. |
PDF Full Text Request