Periplogenin‐3‐O‐ ‐D‐Glucopyranosyl ‐(1→6)‐ ‐D‐Glucopyaranosyl‐ ‐(1→4) ‐D‐Cymaropyranoside,Isolated from Aegle marmelos Protects Doxorubicin Induced Cardiovascular Problems and Hepatotoxicity in Rats

Doxorubicin is a common chemotherapeutic anticancer drug. Its use is associated with adverse effects including cardiotoxicity. Several therapeutics interventions have been attempted to reduce the toxicity and to improve the efficacy of the drug. However, on phytochemicals very few investigations have been made. In the present study we have evaluated the potential of a cardenolide, periplogenin, isolated from the leaves of Aegle marmelos in protecting the doxorubicin induced cardiotoxicity and lipid peroxidation (LPO) in rats. Doxorubicin induced cardiac and hepatotoxicity were characterized by marked biochemical changes including an increase in serum creatine kinase–MB (CK–MB), glutamate‐pyruvate transaminase (SGPT), and tissue LPO, with a decrease in superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH). It also increased the levels of different serum lipids, but decreased the amount of high‐density lipoprotein (HDL). Cotherapy of the test cardenolide and doxorubicin for 4 weeks reversed all these adverse effects. However, out of three different concentrations (12.5, 25, and 50 mg/kg p.o.) of the test periplogenin, 25 mg/kg appeared to be most effective. When its efficacy was compared with that of vitamin E (α‐tocopherol) the isolated compound exhibited a better therapeutic potential. The isolated periplogenin from the leaves of A. marmelos could potentially inhibit doxorubicin‐induced cardiovascular problems in rats. However, its moderate dose was found to be most effective.     

Analysis of N1‐acetyl‐N2‐formyl‐5‐methoxykynuramine/N1‐acetyl‐5‐methoxy‐kynuramine formation from melatonin in mice

Abstract: The interactions of melatonin, a potent endogenous antioxidant, with reactive oxygen species generate several products that include N¹‐acetyl‐N²‐formyl‐5‐methoxykynuramine (AFMK) and N¹‐acetyl‐5‐methoxy‐kynuramine (AMK). The physiological or pathological significance of AFMK/AMK formation during the process of melatonin metabolism in mammals has not been clarified. Using a metabolomic approach in the current study, the AFMK/AMK pathway was thoroughly investigated both in mice and humans. Unexpectedly, AFMK and AMK were not identified in the urine of humans nor in the urine, feces or tissues (including liver, brain, and eyes) in mice under the current experimental conditions. Metabolomic analysis did identify novel metabolites of AMK, i.e. hydroxy‐AMK and glucuronide‐conjugated hydroxy‐AMK. These two newly identified metabolites were, however, not found in the urine of humans. In addition, oxidative stress induced by acetaminophen in the mouse model did not boost AFMK/AMK formation. These data suggest that AFMK/AMK formation is not a significant pathway of melatonin disposition in mice, even under conditions of oxidative stress.     

Expression of 8‐hydroxy‐2′‐deoxyguanosine in chronic liver disease and hepatocellular carcinoma

Abstract: Reactive oxygen species may be involved in the progression of chronic liver disease and the occurrence of hepatocellular carcinoma (HCC). To clarify whether clinicopathological findings in liver diseases are related to oxidative DNA damage, hepatic expression of the 8‐hydroxy‐2′‐deoxyguanosine (8‐OHdG) was examined in 75 liver disease patients, which included 32 chronic hepatitis (CH), 13 liver cirrhosis (LC) and 30 HCC patients. The CH patients had higher 8‐OHdG‐positive hepatocytes than LC (P<0.05). In CH and LC, the number of 8‐OHdG‐positive hepatocytes was correlated with alanine aminotransferase and asparate aminotransferase (P<0.01 and P<0.05, respectively). Of 30 HCC cases, 25 cases (83%) showed stronger immunoreactivity than non‐cancerous counterparts. The patients with poorly differentiated HCC had a larger tumor size and higher levels of AFP, and exhibited higher labeling indices of PCNA‐, TUNEL‐ and 8‐OHdG‐positive cells than those with well and moderately differentiated HCC. Our findings suggest that oxidative DNA damage is increased in association with necroinflammation in chronic liver disease and determination of 8‐OHdG is useful in assessing high‐grade malignancy in HCC.     

2‐Amino‐N‐(2,6‐dichloropyridin‐3‐yl)acetamide derivatives as a novel class of HBV capsid assembly inhibitor

Capsid structure is crucial for the maturation and maintenance of the stable hepatitis B virion. Therefore, chemicals that inhibit capsid assembly might potentially act as potent antiviral compounds. However, only a few chemicals are known to block the capsid assembly process and further viral proliferation. In this study, we present a novel family of capsid assembly inhibitors that act against hepatitis B virus (HBV). Based on X‐ray crystallographic data of the HBV core protein (Cp), we built dimer and hexamer structural models to be used in library searches. Several chemicals in the 2‐amino‐N‐(2,6‐dichloropyridin‐3‐yl)acetamide family were predicted to have high affinity for the groove structure in Cp. Using in vitro assembly and the HepG2.2.15 cell culture test, we verified that these chemicals demonstrated inhibitory effects on capsid assembly. Furthermore, we investigated the combinatorial effects of these assembly inhibitor chemicals with lamivudine and revealed that, in combination, they have synergistic inhibitory effects on decreasing viral concentration. We propose that these inhibitors could be utilized as an effective combination treatment against HBV infection.     

Effect of genipin on cholestasis induced by estradiol‐17β‐glucuronide and lithocholate‐3‐O‐glucuornide in rats

Aim: Genipin is reported to stimulate the insertion of multidrug resistance protein 2 (Mrp2) in the bile canalicular membrane, thereby causing choleresis by the increased the biliary excretion of glutathione, which has been considered to be a substrate of Mrp2. In the present study, we examined the effect of genipin on cholestasis induced by estradiol‐17β‐glucuronide and lithocholate‐3‐O‐glucuronide, Mrp2 substrates, in rats. Further, the effect of genipin on the biliary excretion of substrates of P‐glycoprotein (P‐gp), vinblastine and erythromycin, was also studied. Methods: The effect of genipin infusion at the rate of 0.5 µmol/min/100 g on cholestasis induced by estradiol‐17β‐glucuronide (0.075 µmol/min/100 g for 20 min) and lithocholate‐3‐O‐glucuronide (0.15 µmol/min/100 g for 40 min) was studied. The effect of genipin infusion on the biliary excretion of a tracer dose of vinblastine and erythromycin infused at the rate of 0.1 µmol/min/100 g was also studied. Results: Genipin relieved estradiol‐17β‐glucuronide‐induced cholestasis, and cumulative biliary estradiol‐17β‐glucuronide excretion for 120 min was increased from 50 ± 20%–81 ± 20% dose. In contrast, genipin had no effect on lithocholate‐3‐O‐glucuronide‐induced cholestasis. Biliary excretion of a tracer dose of vinblastine and the maximum biliary excretion of erythromycin were significantly decreased by genipin. Conclusions: Genipin protected estradiol‐17β‐glucuronide‐induced cholestasis. The mechanism of the protection of cholestasis by genipin is unknown, but it is speculated to be due to a conformational change of P‐gp by genipin, in addition to the stimulation of Mrp2 insertion into the bile canaliculi.     

Effects of 1‐O‐hexyl‐2,3,5‐trimethylhydroquinone on carbon tetrachloride‐induced hepatic cirrhosis in rats

Aim: The present study was undertaken to evaluate the effects of 1‐O‐hexyl‐2,3,5‐trimethylhydroquinone (HTHQ), a synthesized vitamin E derivative, on carbon tetrachloride (CCl₄)‐induced cirrhosis. Methods: Rats were treated with hypodermic injections of CCl₄ twice a week to induce the hepatic cirrhosis, and given drinking water containing HTHQ or solvent. Primary cultures of rat hepatocytes were performed to evaluate the effects of HTHQ on the expression of inducible nitric oxide synthase (iNOS). Results: Masson's staining of rat livers showed fibrosis around pseudo‐lobules in the CCl₄ group, the lesions being reduced in the CCl₄ HTHQ group. Increases in liver tissue hydroxyproline and α₁(I) collagen, α‐smooth muscle actin and iNOS induced by CCl₄, were also markedly diminished by HTHQ. Furthermore, both HTHQ and vitamin E attenuated interleukin‐1β‐induced iNOS protein expression in cultured hepatocytes, the potency of HTHQ being 10‐times higher than that of vitamin E. Conclusion: HTHQ may inhibit development of hepatic cirrhosis in rats, more potently than vitamin E, by inhibiting the iNOS expression in hepatocytes. Because vitamin E has a radical scavenging action, roles of NO and peroxynitrite will be discussed in the effects of HTHQ on the fibrosis.