Oxidative stress leading to the formation of free radicals has been implicated in many biological processes, damaging the cell membrane and biological molecules. Free radicals are also responsible for the development of many diseases such as arteriosclerosis, cancer, inflammation, cardiovascular disorder, ischemia, arthritis, and liver diseases. Lipid peroxidation is initiated by very potent free radicals including superoxide (O2) and hydroxyl radicals (OH-). Most of free radicals can be scavenged by the endogenous antioxidant defense systems such as superoxide dismutase (SOD), the glutathione peroxidase/glutathione system, catalase, and peroxidase. But these systems are not always completely efficient, making it imperative to receive exogenous antioxidants originally from the medicinal herbs and diet. Oxidative modification of low-density lipoprotein (LDL) has been suggested to play an important role in the development of human atherosclerosis. Accordingly, protection of LDL from oxidation can retard or effectively prevent the progression of the disease.
There has been an interest over the years in the pronounced free-radical-scavenging activity of phenylethanoid glycosides ascertained both in vitro and in vivo in the stable free-radical systems. An earlier report claimed that phenylethanoid glycoside acteoside showed its antioxidant activity both as a scavenger of superoxide anions generated in a phenazin methosulfate-NADH system and as an inhibitor of lipid peroxidation in mouse liver microsomes (6). In addition, acteoside was found to significantly repair the oxidized OH adducts of 2'-deoxyadenosine-5'-monophosphate acid (dAMP) and 2'-deoxyguanosine-5'-monophosphate acid (dGMP). This implies that it has potent antioxidant activity for reducing the oxidized OH adducts (7-9).
Another group reported that acteoside and its isomer isoacteoside, both having four phenolic hydroxy groups, could strongly protect red blood cells from oxidation-induced hemolysis (10). In antioxidative assays based on 1,1-
diphenyl-2-dipicrylhydrazyl free-radical (DPPH) scavenging and on Trolox equivalent antioxidant activity (TEAC), acteoside and isoacteoside were shown to have free-radical-scavenging effects (11,12).
Furthermore, it was observed that acteoside and isoacteoside had antioxidative effects on lipid peroxidation induced by FeSO4-edetic acid in linoleic acid and on the chelating activity for Fe2 + . The chelating activity for Fe2+ of isoacteoside was twofold stronger than that of acteoside. The inhibitory effects of the two compounds with phenolic hydroxy groups on lipid peroxidation are due to the chelating property. Under physiological logical conditions the phenylethanoid glycoside-Fe2 + chelates are confirmed to be sufficiently stable. Thus the phenylethanoid glycosides are able to inhibit Fe2+-dependent lipid peroxidation by chelating Fe2 + , and their therapeutic potentials are thought to be based on the same mechanism (13).
In addition, antioxidative effects have been observed for 2'-acetylacteo-side, poliumoside, and brandioside isolated from B. hancei. They were shown to have inhibitory effects on free-radical-induced hemolysis of red blood cells and free-radical-scavenging activities in vitro. Brandioside and poliumoside exhibited stronger antioxidant effect than acteoside, 2'-acetylacteoside, and Trolox (14). On the other hand, acteoside, isoacteoside, and 2'-acetylacteo-side had stronger free-radical-scavenging activities than a-tocopherol on DPPH radical and xanthine/xanthine oxidase (XOD)-generated superoxide anion radical. Among the three compounds, isoacteoside with its caffeoyl moiety at the 6'-position of the glucose chain showed an inhibitory effect on XOD. Further studies disclosed that each of them exhibited significant inhibition of both ascorbic acid/Fe2 + - and ADP/NADPH/Fe3 + -induced lipid peroxidation in rat liver microsomes (5,15). Acteoside and isoacteoside had strong protective effects against the oxidation of human LDL from Cu2+-mediated oxidation. They were also effective in preventing the peroxyl free-radical-induced oxidation of a-tocopherol in human LDL. Inhibition of these phenylethanoid glycosides on the oxidation of human LDL and a-tocopherol is dose-dependent in the concentration range of 5-40 ||M (16).
Interestingly, the observed antioxidative effects of phenylethanoid glycosides were found to be dependent of the number of phenolic hydroxyl groups they have. Those with four phenolic hydroxy groups have stronger antioxidative effects than those with only two or less. As to the antioxidative mechanism, these compounds were shown to have at least two mechanisms of scavenging free radicals: they are able to suppress free-radical processes at two stages: the formation of superoxide anions and the production of lipid peroxides. The antioxidative effects would offer a plausible explanation for the observed therapeutic effects for arteriosclerosis. On the other hand, phenyl-ethanoid glycosides in B. hancei may partly account for its ethnomedicinal application for the relief of hyperlipemia and hypercholesterolemia.
In addition, acteoside was shown to decrease significantly the concentration of oxygen free radicals (OFR) and lipid peroxidation in skeletal muscle resulting from exhaustive exercise. The effect of reducing oxidative stress is attributable to decreasing the concentration of free radicals and the level of lipid peroxidation (17). Other investigation demonstrated that acteoside at 20.0 |aM resisted significantly Bufo gastrocnemius muscle fatigue allowed electrically. This observation was attributed to the antioxidative activity of the glycoside, which is in agreement with the role of reactive oxygen species (ROS) in promoting fatigue in skeletal muscle (18).
The use of nonsteroidal antiinflammatory drugs (NSAIDs) is the main therapeutic approach for the treatment of inflammatory diseases, in spite of their renal and gastric side effects. Accordingly, there is a growing need for new anti-inflammatory compounds. Several research groups have particularly focused on searching for anti-inflammatory natural compounds.
Some phenylethanoids have been previously shown to be anti-inflammatory. Acteoside had inhibitory effects on arachidonic-acid-induced mouse ear and carrageenin-induced rat ankle edema when administered orally to the test animals (19,20). Acteoside also inhibits histamine- and bradykinin-induced contractions of guinea pig ileum. These results indicate that acteoside has anti-inflammatory properties that seem to be due, at least partly, to its action on histamine and bradykinin.
Pathogenically, inflammation is a complex process characterized by the involvement of several mediators, including prostaglandins (PGs) and nitric oxide (NO). The pivotal role of PGs in inflammation was firmly established with the discovery that the anti-inflammatory action of drugs was mediated by inhibition of the enzyme cyclooxygenase (COX), which converts arachidonic acid to PGs. Biochemically COX has two isoforms, COX-1 and COX-2. COX-1 is expressed constitutively in most mammalian cells and regulates many physiological functions through the release of PGs, while COX-2 is expressed at a very low level in most tissues, but much higher at the inflammation site. COX-2 contributes to the development of inflammation, suggesting that selective inhibition of the COX-2 isoform could be relevant to the discovery of anti-inflammatory drugs devoid of the typical side effects of most of the traditional NSAIDs (21). NO is a free radical produced in mammalian cells constitutively or induced by various cell activators through the oxidation of L-arginine by a family of isoenzymes known as nitric oxide synthases (NOS). Inhibition of NOS is effective in reducing NO generation and therefore helpful in the treatment of inflammation.
In vitro screening for anti-inflammatory phenylpropanoid glycosides as inhibitors of COX-2 and NO biosynthesis demonstrated that acteoside and arenarioside exert their anti-inflammatory action through the inhibition of COX-2 enzyme to prevent proinflammatory PG generation, as they have greater inhibitory potency on COX-2 than on Cox-1 (22).
In the same experimental model, isoacteoside, acteoside, and 2'-O-acetylacteoside were also found to be inhibitors of NO production. They substantially reduced nitrite accumulation in lipopolysaccharide (0.1 Ag/mL)-stimulated J774.1 cells at the concentration of 100-200 aM. Specically, they inhibited at 200 aM by 32.2-72.4% nitrite accumulation induced by lipopolysaccharide (0.1 Ag/mL)/interferon-g (100 U/mL) in mouse peritoneal exudates macrophages. However, these compounds did not affect the expression of inducible nitric oxide (iNOS) mRNA, the iNOS protein level, or the iNOS activity in lipopolysaccharide-stimulated J774.1 cells. Instead, they had a clear scavenging effect (6.9-43.9%) even at low concentrations (2-10 aM) on nitrite generated from an NO donor, 1-propanamine-3-hydroxy-2-nitroso-1-propylhydrazino (PAPA NONOate). These results indicate that the phenyl-ethanoids have NO radical-scavenging activity, which possibly contributes to their anti-inflammatory effects (23).
Acteoside was also found to be able to induce proinflammatory cytokines interleukin (IL)-1, IL-6, and tumor necrosis factor-a (TNF-a) in macrophage-like cell line J774.A1 at 1-100 ng/mL. Moreover, the stimulatory action of acteoside was studied using the bovine glomerular endothelial cell line GEN-T and it was found that it can stimulate IL-6 production. These stimulatory activities cannot be abolished by treating with polymyxin B, which is capable of inactivating lipopolysaccharide (LPS), indicating thereby that the action was not a contamination of LPS (24).
Antioxidants have been shown to inhibit both initiation and promotion in carcinogenesis and counteract cell immortalization and transformation. Caffeoylated phenylethanoid glycosides acteoside and isoacteoside were cytotoxic and cytostatic, displaying in vivo anticancer activity against murine P-388 (PS) lymphocytic leukemia. The ED50 values were found to be 2.6 Ag/mL for the former and 10 Ag/mL for the latter (25). Acteoside had cytotoxic and cytostatic activity against several kinds of cancer cells. However, they did not affect the growth and viability of primary-cultured rat hepatocytes. Attention to the structure-activity relationship demonstrated that the effects of the compounds appear to be due to their ortho-dihydroxylated aromatic systems (26). They also selectively inhibited the growth of murine melanoma B16F10 cells with the same IC50 value of 8 |aM. Comparison of the action with inhibitory activities of their meth-anolysis products showed that the 3,4-dihydroxyphenethyl alcohol group in acteoside and isoacteoside might be more essential for the activity than the caffeoyl group (27).
Acteoside remarkably decreased the growth curve and mitotic index of human gastric adenocarcinoma MGc80-3 cells and delayed the cell-doubling time in vitro. There was a 75% decrease of the tumorigenicity of the treated cells compared to the untreated cells inoculated subcutaneously in BALB/C nude mice. Scanning electron microscopy revealed that the microvilli on the surface of treated cells had been reduced. It has been confirmed that acteoside could reverse MGc80-3 cells' malignant phenotypic characteristics and induced redifferentiation of MGc80-3 cells (28).
Lung cancer is the third most common cancer in the United States and the leading cause of cancer death. The mortality is high because systemic therapies do not cure metastatic disease. The side effects and the development of drug resistance colimit the use of conventional cytotoxic chemotherapeutic agents for treating patients with lung cancer. An increase in the expression of COX-2 may play a significant role in carcinogenesis in addition to its well-known involvement in the inflammatory reaction (29,30) that is also frequently noted in a specific type of lung cancer. Acteoside, with COX-2 inhibitory and cytotoxic activities, had suppressive effect on lung metastasis of B16 melanoma cells. At a dose of 50 mg/kg acteoside was administered intraperitoneally every other day from 13 days before B16 melanoma cell injection until all mice had succumbed to the lung metastatic tumor burden in the lung. Administration of acteoside significantly prolonged survival time; the average survival time was 63.3 ± 3.4 days compared with 52.1 ± 2.5 days in control mice. The results suggest that effects of acteoside may be involved in the therapeutic effect on lung cancer (31).
Apoptosis is closely related to the development and homeostasis of normal tissues. It has become evident that alterations in the apoptotic pathways are intimately involved in a variety of cancer processes. The mechanism of cell death mediated by cytotoxic chemotherapy was once thought to be through irreversible DNA damage with subsequent mitotic failure. The spectrum of chemotherapeutic agents stimulating apoptosis suggests that the programmed cell death pathway is a central mechanism of the cytotoxic effects of current therapy. Acteoside can induce cell death in promyelocytic leukemia HL-60 cells with an IC50 value of 26.7/|aM. Analysis of extracted DNA on agarose gel electrophoresis revealed that acteoside induced the internucleosomal breakdown of chromatin DNA characteristic of apoptosis. Apoptosis-specific DNA fragmentation was clearly detectable 4 hr after treatment with acteoside and was independent of the cell cycle phase. These data indicate that acteoside induces apoptosis in HL-60 cells (32).
Protein kinase C (PKC) represents a family of more than 11 phospho-lipid-dependent ser/thr kinases that are involved in a variety of pathways. They regulate various celluar processes including mitogenesis, cell adhesion, apoptosis, angiogenesis, invasion, and metastasis (33-36). PKCs are the major cellular targets that can be activated by tumor-promoting phorbol ester, and consequently are thought to play an important role in carcinogen-esis (37). Thus PKCs may be important for both oxidant-mediated tumor promotion and antioxidant-mediated chemoprevention. Conceivably consistent with the generalization that diverse tumor promoters are oxidants, a variety of structurally related chemopreventive agents are antioxidants. These facts demonstrate the functional significance of antioxidant activity in cancer prevention and inhibition. Acteoside, with antioxidant activity, is a potent inhibitor of PKC from the rat brain. Biochemically acteoside, interacting with the catalytic domain of PKC, is a competitive inhibitor with the substrate ATP and a noncompetitive inhibitor with respect to the phosphate acceptor (histone IIIS). This effect was further evidenced by the fact that acteoside inhibited native PKC with an identical catalytic fragment. However, it did not affect (3H)-phorbol-12,13-dibutyrate binding to PKC (38). In accordance with the above finding, it was observed that acteoside, and poliumoside showed inhibitory activity against PKC-a with IC50 values of 9.3 and 24.4 |M, respectively (39). These results suggested that phenolic antioxidants, being easily converted to their oxidized state, may be inhibitory against tumor promotion.
Acteoside and arenarioside are capable of scavenging reactive oxygen species such as superoxide anion, peroxide hydrogen, hypochlorous acid, and hydroxyl radical. Moreover, the use of different stimuli having various pathways of action on polymorphonuclear neutrophils (PMN) oxidative metabolism permits the establishment of the hypothesis that each phenyl-propanoid ester has its own particular mechanism of action through protein kinase C or phospholipase C pathways (40).
In recent years, telomerase has emerged as a highly promising novel target for therapeutic intervention in the treatment of cancer. In approximately 85% of human cancers, however, telomerase is reactivated and acts to maintain telomere length. Acteoside has been identified as a potent inhibitor of telomerase in the human gastric carcinoma cells MKN45. Modeling and biophysical studies suggest that acteoside-mediated cell differentiation and apoptosis may be based on telomere-telomerase-cell-cycle-dependent modulation. Thus, the antitumor mechanism of acteoside is demonstrated once more to be due to its inhibition of telomerase in tumor cells (41).
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