Ampelopsis brevipedunculata (Vitaceae) extract inhibits a progression of carbon tetrachloride-induced hepatic injury in the mice.

Ampelopsis brevipedunculata (Maxim.) Trautv. (Vitaceae) has been used in Japanese herbal folk medicine to treat liver disease. The objective of this study is to evaluate the antihepatotoxic effect of A. brevipedunculata in the mice. An aqueous fraction was extracted by immersing the berries of the plant material in 40% ethanol for six months, followed by removing ethanol. Daily free access to the aqueous extract as drinking water greatly reduced the severity of hepatic injury, characterized by centrilobular necrosis, cytoplasmic vacuolation, cellular swelling, inflammation, and fibrosis in the mice receiving a nonlethal dose of carbon tetrachloride twice weekly during nine weeks. In addition, such a feeding regimen decreased the elevated levels of plasma glutamate oxaloacetate transaminase and glutamate pyruvate transaminase in the carbon tetrachloride-administered mice. These results suggest that the feeding regimen with A. brevipedunculata extract inhibited a progression of hepatic injury induced by carbon tetrachloride.

An ethanol-extract of Ampelopsis brevipedunculata (Vitaceae) berries decreases ferrous iron-stimulated hepatocyte injury in culture.

We characterized the effects of an ethanol-extract of the berries of Ampelopsis brevipedunculata (Maxim.) Trautv. (Vitaceae), a plant used in folk medicine to treat liver disease, on rat hepatocyte injury occurring spontaneously, stimulated with ferrous iron and with xanthine oxidase in combination with hypoxanthine or stimulated with ethanol in serum-free culture. Total intracellular and extracellular activities of lactate dehydrogenase (LDH) accumulating during incubation and the percentage of intracellular LDH activity released into culture medium were routinely measured, to evaluate the degree of the injury. The extract decreased a high level of LDH release spontaneously occurring and an elevated level of LDH release stimulated with ferrous iron to approximately the level caused by antioxidants, such as superoxide dismutase, pyruvate and dimethyl sulfoxide. Xanthine oxidase-stimulated LDH release was not decreased by the extract. Ethanol-stimulated LDH release was decreased by the extract when the spontaneous release level was comparatively high. These results indicate that the extract inhibits intact hepatocytes from degrading, by the toxic effect of iron released from primary injured hepatocytes through the generation of reactive oxygen species. The major antitoxic activity of the extract was found in an undialyzable fraction. Sugars were necessary to exert the activity as estimated by periodate oxidation of the extract.

Effects of iron chelates on the transferrin-free culture of rat dermal fibroblasts through active oxygen generation.

Effects of nonchelating and chelating agents at 10 mM on the serum-free culture of rat dermal fibroblasts were investigated. A strong iron-chelating agent, iminodiacetic acid (IDA), and a weak one, dihydroxyethylglycine (DHEG), decreased iron permeation into preconfluent fibroblasts. A weak iron-chelating agent, glycylglycine (GG), a nonchelating agent, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), and human apotransferrin (10 micrograms/ml) increased the permeation with time. Iron may be essential for survival of fibroblasts because subconfluent fibroblasts exposed to 100 microM FeSO4 in combination with transferrin, HEPES, or GG significantly decreased to release lactate dehydrogenase into the medium. Superoxide dismutase and dimethyl sulfoxide blocked the enzyme release, suggesting that superoxide and hydroxyl radical induce cellular damage but hydrogen peroxide (H2O2) generated by superoxide dismutation does not. GG significantly reduced H2O2 cytotoxicity. DHEG acted as a potent promoter of the iron-stimulated cellular damage if ascorbate or H2O2 was added to the medium. FeSO4 and FeCl3 (50 to 100 microM) individually combined with IDA maximally promoted fibroblast proliferation. Ascorbate increased formation of thiobarbituric acid-reactive substances from deoxyribose in the medium supplemented with FeSO4 and either IDA or DHEG. Conversely, ascorbate decreased the formation in the medium with FeSO4 and with or without other agents. Fibroblast proliferation may thus be stimulated through the active oxygen generation mediated by a redox-cycling between Fe3+ and Fe2+, which are dissolved in the medium at a high concentration, rather than through delivery of iron into the cells.

Effects of Ampelopsis brevipedunculata (Vitaceae) extract on hepatic M cell culture: function in collagen biosynthesis.

A spirits-extracted fraction of berries of Ampelopsis brevipedunculata (Maxim.) Trautv. (Vitaceae) is used in Japanese folk medicine to treat liver disease. Since such an extract has been shown to inhibit formation of collagen fibers by rat hepatic M cells, it was felt that the extract acts as an inhibitor of hepatic fibrosis. Amino acid analysis of fibrous substances developing on M cell layers and of a cell lysate fraction indicated that an A. brevipedunculata extract inhibited collagen formation. Biosynthesis of non-collagenous proteins and collagen was evaluated by measuring the extent of [3H]tryptophan incorporation into a protein fraction and the rate of [3H]proline incorporation into a collagenase-digestible fraction, respectively. In contrast to the results of the analysis of the fibrous substances, the A. brevipedunculata extract failed to decrease synthesis of non-collagenous proteins and collagen unless cell proliferation was inhibited. There was no detectable level of collagenolytic activity in the M cell culture with the A. brevipedunculata extract. The decrease in accumulation of collagen, therefore, appeared to be a consequence of the proliferation-inhibitory effect of the A. brevipedunculata extract. Such inhibitory activity was found in a macromolecular fraction that contained abundant sugars but lacked proteins.

Ampelopsis brevipedunculata (Vitaceae) extract stimulates collagen synthesis through superoxide generation in the serum-free cultures of rat dermal fibroblasts and Ito cells.

We describe the effects of an ethanol-extracted fraction of berries of Ampelopsis brevipedunculata (Maxim.) Trautv. (Vitaceae), a plant used in folk medicine to treat liver disease, on the synthesis of non-collagenous proteins and collagen by rat collagen-producible cells such as dermal fibroblasts and liver non-parenchymal Ito cells. The generation of superoxide and hydroxyl radical was assessed by measuring the reduction of cytochrome c and the formation of thiobarbituric acid-reactive substances from deoxyribose, respectively. The synthesis of non-collagenous proteins and collagen as evaluated by measuring the extent of [3H]tryptophan incorporation into a total protein fraction of culture products and the [3H]proline-incorporating rate into a collagenase-digestible protein fraction, respectively. Both types of cells promptly synthesized only collagen in response to a dialyzable fraction of the extract. Major activity to generate oxygen free radicals accumulated in the dialyzable fraction whereas activity to decrease ferrous iron-mediated generation of the radicals accumulated in an undialyzable fraction of the extract. Stimulation of collagen synthesis was caused by superoxide because addition of superoxide dismutase but not pyruvate, an antioxidant of hydrogen peroxide, or dimethyl sulfoxide, an antioxidant of the hydroxyl radical, abrogated the stimulatory effect. The extract may arrest the progress of liver injury mediated by oxygen free radicals generated in the presence of ferrous iron.

Role of iron chelators in growth-promoting effect on mouse hybridoma cells in a chemically defined medium.

The role of various iron chelators on the multiplication of mouse hybridoma cells in an albumin-free, transferrin-deficient defined medium was investigated. Fe(III)-dihydroxyethylglycine, Fe(III)-glycylglycine, Fe(III)-ethylenediamine-N,N'-dipropionic acid, or Fe(III)-iminodiacetic acid supported the excellent growth of the cells. In addition, the growth of the iron-starved cells, which had been preincubated in a protein-, iron- and chelator-free defined medium, restored rapidly when the medium was supplemented with holotransferrin, ferric iron, and chelator compared to that when supplemented with holotransferrin, but without iron and chelator. The results suggest that such chelators modulate a progression of transferrin cycle in the presence of transferrin and ferric iron. An alternative explanation is that there is a decrease in generation of iron-catalyzed free radicals.

Effects of ceruloplasmin and the haptoglobin-hemoglobin complex on the oxygen-dependent reduction in growth of human diploid fibroblasts in serum-free, albumin containing medium.

We examined that growth-promoting activity of two different human albumin (HSA) preparations for human diploid fibroblasts in serum-free RITC 80-7 medium. The activity of one preparation (sample A) was affected markedly by environmental oxygen, whereas the other (sample B) was little affected. Sample B contained ceruloplasmin (Cp) and haptoglobin (Hp) as impurities. To detect the generation of superoxide anion in the media the amount of reduction of cytochrome c that is inhibited by superoxide dismutase (SOD) was determined. In an aerobic environment it was relatively large in comparison with reduction inhibited in a hypoxic environment. Reduction in the sample A with HSA-supplemented medium was relatively large in comparison with that in sample B with HSA-supplemented medium. The reduction of cytochrome c also was inhibited by Cp (25 mg/l) and catalase (4000 units/ml). Moreover, SOD, Cp, catalase and Hp.Hb (but not Hp) partially prevented oxygen-dependent reduction in growth in an aerobic environment when added to sample A HSA-supplemented medium. These results suggest that Cp and Hp.Hb act as an antioxidants in culture.