Antioxidant property of an active component purified from the leaves of paraquat-tolerant Rehmannia glutinosa

Abstract

Acteoside extracted from the leaves of Rehmannia glutinosa was examined to determine the mechanism(s) of its antioxidant properties. The deoxyribose assay system showed that acteoside has a high redox potential as electron donor, which generates hydroxyl radicals in an Fe³⁺-dependent manner similar to ascorbic acid. However, the antioxidant properties of acteoside differ from those of ascorbic acid in that the superoxide anion-mediated reduction of nitroblue tetrazolium was actively inhibited by acteoside but not by ascorbic acid. Acteoside protected cells against glucose oxidase-mediated cytotoxicity and apoptosis in a dose-dependent manner. In addition, acteoside had immune stimulating effects, as shown by the acteoside-mediated increase in the level of DNA synthesis, viability, and cytokine secretion in mouse splenocytes. Moreover, acteoside inhibited the gelatinolytic activity of MMP proteins in a dose-dependent manner. Considering these results and the fact that acteoside is a water-soluble natural product, acteoside might have potential as a preventative treatment for oxidative stress-mediated diseases and have possibilities in the cosmetic industry.     

Acteoside and its aglycones protect primary cultures of rat cortical cells from glutamate-induced excitotoxicity

We have previously reported that acteoside isolated from the leaves of Callicarpa dichotoma has significant neuroprotective activity against glutamate-induced neurotoxicity in primary cultured rat cortical cells. To determine the essential structural moiety within this phenylethanoid glycoside needed to exert neuroprotective activity, acteoside was hydrolyzed with acid into its aglycones, caffeic acid and 3',4'-dihydroxylphenylethanol. Caffeic acid and 3',4'-dihydroxylphenylethanol also showed significant neuroprotective activities. Acteoside and its aglycones inhibited glutamate-induced intracellular Ca2+ influx resulting in overproduction of nitric oxide and reduced the formation of reactive oxygen species. These compounds preserved the mitochondrial membrane potential and the activities of antioxidative enzymes, such as superoxide dismutase, glutathione reductase and glutathione peroxidase reduced by glutamate. It was followed by the preservation of the level of glutathione and finally the inhibition of membrane lipid peroxidation.     

Protective effect of acteoside on carbon tetrachloride-induced hepatotoxicity

This study investigated the protective effects of acteoside, a phenylethanoid glycoside, on the carbon tetrachloride-induced hepatotoxicity as well as the possible mechanisms involved in this protection in mice. Pretreatment with acteoside prior to the administration of carbon tetrachloride significantly prevented the increased serum enzymatic activities of alanine and aspartate aminotransferase in a dose-dependent manner. In addition, pretreatment with acteoside significantly prevented the increase in hepatic malondialdehyde formation and the depletion of the reduced glutathione content in the liver of carbon tetrachloride-intoxicated mice. Carbon tetrachloride-induced hepatotoxicity was also essentially prevented, as indicated by a liver histopathologic study. The effects of acteoside on cytochrome P450 (P450) 2E1, the major isozyme involved in carbon tetrachloride bioactivation were also investigated. Treatment of the mice with acteoside resulted in a significant decrease in the P450 2E1-dependent pnitrophenol and aniline hydroxylation in a dose-dependent manner. Consistent with these observations, the P450 2El protein levels were also lower. Acteoside exhibited anti-oxidant effects on FeCl2-ascorbate induced lipid peroxidation in a mouse liver homogenate, and on superoxide radical scavenging activity. These results suggest that the protective effects of acteoside against the carbon tetrachloride-induced hepatotoxicity possibly involve mechanisms related to its ability to block the P450-mediated carbon tetrachloride bioactivation and free radical scavenging effects.     

毛蕊花糖苷的生物合成研究进展

毛蕊花糖苷是由咖啡酸、羟基酪醇、葡萄糖和鼠李糖组成的苯乙醇苷类衍生物,广泛存在于多种天然药用植物中,具有抗炎、抗菌、抗病毒、抗肿瘤、抗氧化、镇痛、神经保护、改善性功能、免疫调节和改善细胞记忆等多方面的生物活性.然而,植物提取来源的毛蕊花糖苷存在含量低、环境污染问题,无法实现绿色可持续的规模化生产.利用合成生物学成功构建...     

Acteoside improves survival in cecal ligation and puncture-induced septic mice via blocking of high mobility group box 1 release

Acteoside, an active phenylethanoid glycoside, has been used traditionally as an anti-inflammatory agent. The molecular mechanism by which acteoside reduces inflammation was investigated in lipopolysaccharide (LPS)-induced Raw264.7 cells and in a mouse model of cecal ligation and puncture (CLP)-induced sepsis. In vitro, acteoside inhibits high mobility group box 1 (HMGB1) release and iNOS/NO production and induces heme oxygenase-1 (HO-1) expression in a concentration-dependent manner, while HO-1 siRNA antagonizes the inhibition of HMGB1 and NO. The effect of acteoside is inhibited by the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 and Nfr2 siRNA, indicating that acteoside induces HO-1 via p38 MAPK and NF-E2-related factor 2 (Nrf2). In vivo, acteoside increases survival and decreases serum and lung HMGB1 levels in CLP-induced sepsis. Overall, these results that acteoside reduces HMGB1 release and may be beneficial for the treatment of sepsis.     

Attenuated endothelium-mediated relaxation by acteoside in rat aorta: Role of endothelial [Ca2+]i and nitric oxide/cyclic GMP pathway

Acteoside and other phenylethanoid glycoside are contained in many plants that are widely used in traditional Chinese herbal medicine. Acteoside possesses multiple biological actions. Its effect on the vascular system is, however, incompletely understood. This study was aimed to investigate the role of endothelial [Ca²⁺]_i, nitric oxide (NO), and cyclic GMP in acteoside-induced inhibition of endothelial NO-mediated relaxation in rat aorta. Acteoside reduced endothelial NO-dependent relaxation induced by acetylcholine (Ach) or A23187. Acteoside inhibited Ach-stimulated increase in tissue content of cyclic GMP in endothelium-intact rings. L-NNA abolished the stimulatory effect of Ach. Treatment with acteoside significantly suppressed bradykinin-induced increase in [Ca²⁺]_i of cultured rat aortic endothelial cells. Acute exposure to acteoside (30 μM) did not affect the expression of eNOS mRNA in endothelium-intact rings. In summary, acteoside impairs endothelial NO-mediated aortic relaxation partially through inhibition of agonist-induced endothelial Ca²⁺ mobilization and Ca²⁺-dependent NO production and subsequent suppression of cyclic GMP formation. This novel pharmacological action if occurring in small vessels in vivo, may contribute to the reported anti-inflammatory effect of acteoside against NO-mediated vascular permeability-related acute edema.     

Antioxidant activity of extract of Rhus oxyacantha root cortex

In the present study, the root extract of Rhus oxyacantha contained 25.33 mg of catechin equivalent per mg of fresh wt and was found rich in proanthocyanidins compared to vine shoot, grape pips and leaves. The chromatographic analysis of the extract suggested the presence of (+) catechin, (-) epicatechin -3-O-gallate as well as proanthocyanidinic oligomers and polymers. Root cortex inhibited the ascorbic acid oxidation by dioxygen. It also prevented DDT-induced thymocytes death in a dose-dependent manner. The results suggested antioxidant property of root extract of Rhus oxyacantha which could be ascribed to its free radical scavenging nature.     

Evaluation of antioxidant activity of <Emphasis Type="Italic">Melothria maderaspatana in vitro</Emphasis>

In this study, an aqueous extract of leaves from Melothria maderaspatana was tested for in vitro antioxidant activity. Free radical scavenging assays, such as hydroxyl radical, hydrogen peroxide, superoxide anion radical and 2,2-diphenyl-1-picryl hydrazyl (DPPH), 2,2’-azinobis-(3-ethyl-enzothiazoline-6-sulfonic acid) (ABTS) radical scavenging, and reducing power assay, were studied. The extract effectively scavenged hydroxyl radical, hydrogen peroxide and superoxide anion radicals. It also scavenged DPPH and ABTS radicals. Furthermore, it was found to have reducing power. All concentrations of leaf extract exhibited free radical scavenging and antioxidant power, and the preventive effects were in a dose-dependent manner. The antioxidant activities of the above were compared to standard antioxidants such as butylated hydroxytoluene (BHT), ascorbic acid, and α-tocopherol. The results obtained in the present study indicate that the M. maderaspatana extract could be considered a potential source of natural antioxidant.     

Effect of Escherichia coli endotoxin on ascorbic acid transport in isolated adrenocortical cells

The active uptake of ascorbic acid by isolated rat adrenocortical cells increases with ascorbic acid concentration, depends on time and calcium, and is inhibited by ACTH concentrations required for maximal steroidogenesis. Lipopolysaccharide of Escherichia coli 0111:B4 modifies the ascorbic acid uptake in a calcium-dependent manner. At low calcium concentrations, lipopolysaccharide exerts a stimulatory effect on ascorbic acid transport and at high concentrations lipopolysaccharide produces a dose-dependent inhibitory effect. This inhibition of the ascorbic acid transport by the endotoxin can alter the ascorbic acid accumulation in the adrenal gland during endotoxin shock.     

Stimulatory action of calcium L-threonate on ascorbic acid uptake by a human T-lymphoma cell line

The effects of preincubation of human T-lymphoma cells with increasing concentrations of calcium L-threonate on the uptake of L-[1-14C]ascorbic acid were examined. Calcium L-threonate (0-1,000 mg%) stimulated ascorbic acid (1.25 mg%) uptake in a dose-dependent manner. These results indicate that calcium threonate and possibly other ascorbic acid metabolites have biological activity and potential pharmacological applications.     

Acteoside inhibits apoptosis in D-galactosamine and lipopolysaccharide-induced liver injury.

We assessed the effect of acteoside, a naturally occurring antioxidative phenylethanoid, on hepatic apoptosis and the subsequent liver failure induced by D-Galactosamine (D-GalN) and lipopolysaccharide (LPS). A co-administration of D-GalN (700 mg/kg) and LPS (35 microg/kg) to mice evoked typical hepatic apoptosis characterized by DNA fragmentation and apoptotic body formation, resulting in fulminant hepatitis and lethality of mice. Pre-administration of acteoside at 10 or 50 mg/kg subcutaneously at 12 and 1 h prior to D-GalN/LPS intoxication significantly inhibited hepatic apoptosis, hepatitis and lethality. Tumor necrosis factor-alpha (TNF-alpha) secreted from LPS-stimulated macrophages is an important mediator of apoptosis in this model. Acteoside showed no apparent effect on the marked elevation of serum TNF-alpha, but it partially prevented in vitro TNF-alpha (100 ng/ml)-induced cell death in D-GalN (0.5 mM)-sensitized hepatocytes at the concentrations of 50, 100 and 200 microM. These results indicated that D-GalN/LPS-induced hepatic apoptosis can be blocked by an exogenous antioxidant, suggesting the involvement of reactive oxygen intermediates (ROIs) in TNF-alpha-dependent hepatic apoptosis.     

Determination of acteoside in Cistanche deserticola and Boschniakia rossica and its pharmacokinetics in freely-moving rats using LC-MS/MS

A sensitive LC-MS/MS method with a simple solid-phase extraction for the determination of acteoside in rat plasma and tissue homogenates was established for the investigation of bioavailability and brain distribution in freely-moving rats. Acteoside in Cistanche deserticola and Boschniakia rossica was also determined. Acteoside and internal standard were separated on a RP-select B column (125mmx4.6mm i.d., particle size 5microm). The mobile phase consisted of 35% methanol and 65% acetic acid-water (1:100, v/v) at a flow-rate of 1mL/min. Acteoside and the internal standard were monitored using the multiple-reaction monitoring (MRM) mode at m/z transitions of 623-->161 and 609-->301, respectively. The acteoside content was 38.4+/-2.4mg/kg (n=3) for B. rossica, which is obviously lower than 21134.2+/-805.5mg/kg (n=3) of C. deserticola. The protein binding in rat plasma was 75.5+/-1.8%. The brain distribution result indicated that acteoside was evenly distributed in brain tissues (brain stem, cerebellum, the rest of the brain, cortex, hippocampus and striatum) which was about 0.45-0.68% of that in plasma (4.5+/-0.5microg/mL) after 15min of acteoside administration (10mg/kg, i.v.). After acteoside was given (3mg/kg, i.v.; 100mg/kg, p.o.), the oral bioavailability (AUC(p.o.)/dose(p.o.))/(AUC(i.v.)/dose(i.v.)) was only 0.12%.     

Semax prevents learning and memory inhibition by heavy metals

Separate and joint effect of Semax, ascorbic acid, lead diacetate, and ammonium molybdate on avoidance conditioning in rats was studied. It was established that the heavy metal salts inhibited the avoidance response, and the peptide counteracted this inhibition as strongly as ascorbic acid or to a comparable degree. These findings confirm the antioxidant properties of Semax.     

Liquid chromatographic method with amperometric detection to determine acteoside in rat blood and brain microdialysates and its application to pharmacokinetic study

A simple and sensitive liquid chromatography with amperometric detection was developed for the first time to monitor the protein-unbound acteoside in the rat blood and brain microdialysate by microdialysis technique. Microdialysis samples without further cleanup procedures were directly injected into the HPLC and separated using a reversed-phase C18 column (150 mmx2 mm, i.d. 5 microm) maintained at ambient temperature and a mobile phase comprised of acetonitrile-50 mM monosodium phosphate (pH 2.8) (17:83, v/v) with a flow rate of 0.2 mL/min. Based on the experimental voltamogram, the applied potential was set at +0.9 V oxidative mode. The concentration-response relationship was linear (r2>0.99) over a concentration range of 5-500 ng/mL; method precision and accuracy fell within predefined limits (less than 20%). The developed method was applied to assess the pharmacokinetics of acteoside, and the results suggested that acteoside was fitted better by the two-compartmental model following a single intravenous injection of acteoside. Acteoside was unable to be detected in the brain dialysate. The distribution and elimination half-lives of unbound acteoside in the blood were 5 and 28 min, respectively, which suggested the rapid distribution of acteoside.     

Effect of antioxidant supplementation on the adaptive response of human skin fibroblasts to UV-induced oxidative stress

The effect of supplementation with substances having antioxidant properties on the adaptive responses of human skin fibroblasts to UV-induced oxidative stress was studied in vitro. UVR was found to induce a substantial oxidative stress in fibroblasts, resulting in an increased release of superoxide anions and an increase in lipid peroxidation (shown by an elevated malonaldehyde content). Sub-lethal doses of UVR were also found to induce adaptive responses in the fibroblast antioxidant defences, with a transient rise in catalase and superoxide dismutase activities followed by a slower, large increase in cellular glutathione content. Supplementation of the fibroblasts with the antioxidants, Trolox (a water soluble analogue of alpha-tocopherol), ascorbic acid or beta-carotene, had differential effects on these responses. Trolox supplementation reduced the UVR-induced cellular oxidative stress and adaptive response in a predictable concentration-dependent manner. This was in contrast to ascorbic acid which increased superoxide release from fibroblasts. At low doses, ascorbate supplements also reduced the magnitude of the adaptive increases in catalase and superoxide dismutase activities and increase in glutathione content. Beta-carotene had a similar effect to ascorbic acid, reducing the extent of the adaptations to UVR at lower doses while simultaneously increasing superoxide release and malonaldehyde content. These in vitro data indicate that only the vitamin E analogue suppressed UVR-induced oxidative stress in a predictable manner and suggest that common dietary antioxidants may not be equally effective in reducing the potential deleterious effects of UVR-induced oxidative stress in skin.     

Collagen synthesis is required for ascorbic acid-enhanced differentiation of mouse embryonic stem cells into cardiomyocytes

Ascorbic acid has been reported to promote the differentiation of embryonic stem (ES) cells into cardiomyocytes; however, the specific functions of ascorbic acid have not been defined. A stable form of ascorbic acid, namely, l-ascorbic acid 2-phosphate (A2-P), significantly enhanced cardiac differentiation; this was assessed by spontaneous beating of cardiomyocytes and expression of cardiac-specific markers obtained from mouse ES cells. This effect of ascorbic acid was observed only when A2-P was present during the early phase of differentiation. Treatment with two types of collagen synthesis inhibitors, l-2-azetidine carboxylic acid and cis-4-hydroxy-d-proline, significantly inhibited the A2-P-enhanced cardiac differentiation, whereas treatment with the antioxidant N-acetyl cysteine showed no effect. These findings demonstrated that ascorbic acid enhances differentiation of ES cells into cardiomyocytes through collagen synthesis and suggest its potential in the modification of cardiac differentiation of ES cells.     

Ascorbic acid 6-palmitate suppresses gap-junctional intercellular communication through phosphorylation of connexin 43 via activation of the MEK–ERK pathway

Although the health benefits of dietary antioxidants have been extensively studied, their potential negative effects remain unclear. L-Ascorbic acid 6-palmitate (AAP), a synthetic derivative of ascorbic acid (AA), is widely used as an antioxidant and preservative in foods, vitamins, drugs, and cosmetics. Previously, we found that AA exerted an antitumor effect by protecting inhibition of gap-junctional intercellular communication (GJIC), which is closely associated with tumor progression. In this study, we examined whether AAP, an amphipathic derivative of AA, has chemopreventive effects using a GJIC model. AAP and AA exhibited dose-dependent free radical-scavenging activities and inhibited hydrogen peroxide (H₂O₂)-induced intracellular reactive oxygen species (ROS) production in normal rat liver epithelial cells. Unexpectedly, however, AAP did not protect against the inhibition of GJIC induced by H₂O₂; instead, it inhibited GJIC synergistically with H₂O₂. AAP inhibited GJIC in a dose-dependent and reversible manner. This inhibitory effect was not due to the conjugated lipid structure of AAP, as treatment with palmitic acid alone failed to inhibit GJIC under the same conditions. The inhibition of GJIC by AAP was restored in the presence of mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor U0126, but not in the presence of other signal inhibitors and antioxidant (PKC inhibitors, EGFR inhibitor, NADPH oxidase inhibitor, catalase, vitamin E, or AA), indicating the critical involvement of MEK signaling in the GJIC inhibitory activity of AAP. Phosphorylation of ERK and connexin 43 (Cx43) was observed following AAP treatment, and this was reversed by U0126. These results suggest that the AAP-induced inhibition of GJIC is mediated by the phosphorylation of Cx43 via activation of the MEK–ERK pathway. Taken together, our results indicate that AAP has a potent carcinogenic effect, and that the influence of dietary antioxidants on carcinogenesis may be paradoxical.     

High dose of ascorbic acid induces cell death in mesothelioma cells

Malignant mesothelioma is an asbestos-related fatal disease with no effective cure. Recently, high dose of ascorbate in cancer treatment has been reexamined. We studied whether high dose of ascorbic acid induced cell death of four human mesothelioma cell lines. High dose of ascorbic acid induced cell death of all mesothelioma cell lines in a dose-dependent manner. We further clarified the cell killing mechanism that ascorbic acid induced reactive oxygen species and impaired mitochondrial membrane potential. In vivo experiment, intravenous administration of ascorbic acid significantly decreased the growth rate of mesothelioma tumor inoculated in mice. These data suggest that ascorbic acid may have benefits for patients with mesothelioma.     

Decreased levels of ascorbic acid in lung following exposure to ozone

Mice were exposed to concentrations of 20, 40 and 200 ppm ozone in air for 30 min. Ozone exposure decreased lung ascorbic acid levels and increased lung weight by up to 50% in a dose related manner. On incubation in Krebsphosphate solution, lung slices from mice exposed to 200 ppm ozone released a smaller fraction of their content of ascorbic acid into the medium than did lung slices from control mice, suggesting that there was a preferential loss of extracellular ascorbic acid during ozone exposure. These results are consistent with the proposed function of ascorbic acid as an extracellular antioxidant in lungs.