It has been shown that lipid peroxidation is associated with hepatic fibrosis and stellate cell activation. Shosaiko-to (TJ-9) is an herbal medicine, which is commonly used to treat chronic hepatitis in Japan, although the mechanism by which TJ-9 protects against hepatic fibrosis is not known. As a result, we assayed the preventive and therapeutic effects of TJ-9 on experimental hepatic fibrosis, induced in rats by dimethylnitrosamine (DMN) or pig serum (PS), and on rat stellate cells and hepatocytes in primary culture, and assessed the antioxidative activities and the active components of TJ-9. Male Wistar rats were given a single intraperitoneal injection of 40 mg/kg DMN or 0.5 mL PS twice weekly for 10 weeks. In each model, rats were fed a basal diet throughout, or the same diet, which also contained 1.5% TJ-9, for 2 weeks before treatment or for the last 2 weeks of treatment. TJ-9 suppressed the induction of hepatic fibrosis, increased hepatic retinoids, and reduced the hepatic levels of collagen and malondialdehyde (MDA), a production of lipid peroxidation. Immunohistochemical examination showed that TJ-9 reduced the deposition of type I collagen and the number of ␣-smooth muscle actin (␣-SMA) positive-stellate cells in the liver and inhibited, not only lipid peroxidation in cultured rat hepatocytes that were undergoing oxidative stress, but also the production of type I collagen, ␣-SMA expression, cell proliferation, and oxidative burst in cultured rat stellate cells. In addition, TJ-9 inhibited Fe 2؉ /adenosine 5Ј-diphosphate-induced lipid peroxidation in rat liver mitochondria in a dose-dependent manner and showed radical scavenging activity. Among the active components of TJ-9, baicalin and baicalein were found to be mainly responsible for the antioxidative activity. These findings suggest that Sho-saiko-to (TJ-9) functions as a potent antifibrosuppressant by inhibition of lipid peroxidation in hepatocytes and stellate cells in vivo. (HEPATOLOGY 1999;29:149-160)

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To clarify the metabolic fate of glycyrrhizin when orally ingested, we investigated the bioavailability of glycyrrhetic acid, the aglycone of glycyrrhizin, after intravenous or oral administration of glycyrrhetic acid (5.7 mg kg-1, equimolar to glycyrrhizin) or glycyrrhizin (10 mg kg-1) at a therapeutic dose in rat. Plasma concentration of glycyrrhetic acid rapidly decreased after its intravenous administration, with AUC of 9200 +/- 1050 ng h mL-1 and MRT of 1.1 +/- 0.2 h. The AUC and MRT values after oral administration were 10600 +/- 1090 ng h mL-1 and 9.3 +/- 0.6 h, respectively. After oral administration of glycyrrhizin, the parent compound was not detectable in plasma at any time, but glycyrrhetic acid was detected at a considerable concentration with AUC of 11700 +/- 1580 ng h mL-1 and MRT of 19.9 +/- 1.3 h, while glycyrrhetic acid was not detected in plasma of germ-free rats at 12 h after oral administration of glycyrrhizin. The AUC value of glycyrrhetic acid after oral administration of glycyrrhizin was comparable with those after intravenous and oral administration of glycyrrhetic acid, indicating a complete biotransformation of glycyrrhizin to glycyrrhetic acid by intestinal bacteria and a complete absorption of the resulting glycyrrhetic acid from intestine. Plasma glycyrrhizin rapidly decreased and disappeared in 2 h after intravenous administration. AUC and MRT values were 2410 +/- 125 micrograms min mL-1 and 29.8 +/- 0.5 min, respectively. Plasma concentration of glycyrrhetic acid showed two peaks a small peak at 30 min and a large peak at 11.4 h, after intravenous administration of glycyrrhizin, with an AUC of 15400 +/- 2620 ng h L-1 and an MRT of 18.8 +/- 1.0 h. The plasma concentration profile of the latter large peak was similar to that of glycyrrhetic acid after oral administration of glycyrrhizin, which slowly appeared and declined. The difference of MRT values (19.9 and 9.3 h) for plasma glycyrrhetic acid after oral administration of glycyrrhizin and glycyrrhetic acid suggests the slow conversion of glycyrrhizin into glycyrrhetic acid in the intestine.

The water-soluble extract from a Paraguayan natural medicine, Nangapiry, the leaves of Eugenia uniflora L. (Myrtaceae), which has been used as an antidiabetic agent, was found to show inhibitory activities on the increase of plasma glucose level in the sucrose tolerance test (STT) conducted with mice. The portion adsorbed on a cation exchange resin was also found to inhibit a-glucosidases. From the active portion, two new active compounds named uniflorines A ( 1 ) and B ( 2 ) and known (+)-(3a, 4a, 5ß)-1-methylpiperidine-3, 4, 5-triol ( 3 ) were isolated. The structures of uniflorines A and B were determined as (-)-(1S, 2R, 6S, 7R, 8R, 8aR)-1,2,6,7,8-pentahydroxyindolizidine and (+)-(1S, 2R, 5R, 7R, 8S, 8aS)-1,2,5,7,8-pentahydroxyindolizidine by spectral means, respectively.

Bufalin is the major component of Chan-Su (a traditional Chinese medicine, TCM) extracts from the venom of Bufo bufo gargarizan. In the present study, we investigated the pharmacological mechanisms of cell cycle arrest and autophagic cell death induced by bufalin in SK-HEP-1 human hepatocellular carcinoma cells in vitro. Bufalin inhibited cell survival by MTT assay and increased cell death by trypan blue exclusion assay in a concentration-dependent manner. In addition, bufalin induced G2/M phase arrest by reducing CDK1 activity. Bufalin triggered DNA fragmentation and apoptotic cell death in SK-HEP-1 cells by DNA gel electrophoresis, TUNEL and caspase-3 activity assay, while bufalin induced autophagic cell death by double-membrane vacuoles (transmission electron microscopy, TEM), acidic vesicular organelles (acridine orange staining) and cleavage of microtubule-associated protein 1 light chain 3 (LC3). Protein expression levels of cyclin A and B, CDK1, phospho-CDK1 (Thr161), Cdc25c, phospho-Cdc25c (Ser198), phospho-AKT (Thr308), phospho-AKT (Ser473), phospho‑mTOR (Ser2481) were downregulated. In contrast, protein expression levels of the Chk1, Wee1, LC3-II, Beclin-1, Atg 5, Atg 7 and Atg 12 were upregulated in SK-HEP-1 cells after bufalin treatment. Inhibition of autophagy by 3-methyladenine (an inhibitor of class III phosphatidylinositol-3 kinase; 3-MA) or bafilomycin A1 (an inhibitor of the vacuolar proton pump of lysosomes and endosomes) reduced the effect of bufalin on cell viability and enhanced the effect of bufalin on apoptosis. In conclusion, bufalin triggered autophagic cell death and G2/M phase arrest through the AKT/mTOR signaling pathway in SK-HEP-1 cells. Our findings showed that bufalin may be potentially efficacious in the treatment of human hepatocellular carcinoma.