Tsui-Hwa Tseng

Protective effect of Hibiscus anthocyanins against tert-butyl hydroperoxide-induced hepatic toxicity in rats.

Hibiscus anthocyanins (HAs), a group of natural pigments occurring in the dried flowers of Hibiscus sabdariffa L., which is a local soft drink material and medical herb, were studied for antioxidant bioactivity. The preliminary study showed that HAs were able to quench the free radicals of 1,1-diphenyl-2-picrylhydrazyl. This antioxidant bioactivitiy was further evaluated using the model of tert-butyl hydroperoxide (t-BHP)-induced cytotoxicity in rat primary hepatocytes and hepatotoxicity in rats. The results demonstrated that HAs, at the concentrations of 0.10 and 0.20 mg/ml, significantly decreased the leakage of lactate dehydrogenase and the formation of malondialdehyde induced by a 30-min treatment of t-BHP (1.5 mM). The in vivo investigation showed that the oral pretreatment of HAs (100 and 200 mg/kg) for 5 days before a single dose of t-BHP (0.2 mmol/kg, ip) significantly lowered the serum levels of hepatic enzyme markers (alanine and aspartate aminotransferase) and reduced oxidative liver damage. The histopathological evaluation of the liver revealed that Hibiscus pigments reduced the incidence of liver lesions including inflammatory, leucocyte infiltration, and necrosis induced by t-BHP in rats. Based on the results described above, we speculate that Hibiscus pigments may play a role in the prevention of oxidative damage in living systems.

Inhibition of cell cycle progression in human leukemia HL-60 cells by esculetin

Esculetin, a coumarin compound, was found to inhibit cell growth and cell cycle progression by inducing arrest of the G1 phase in HL-60 cells. To obtain information regarding cell cycle arrest induced by esculetin, we examined its effect on the regulating factors of the G1 phase in the leukemia HL-60 cells treated with esculetin by Western blotting. Our observations were: (1) a distinct increase in the level of hypophosphorylated retinoblastoma protein (pRb), and a reduction in the level of CDK4 after treatment with 100 microM of esculetin for 24 h; (2) a marked up-regulation of p27, and a down-regulation of cyclin D1 after treatment with 100 microM esculetin for 24 h. These results suggest that esculetin can inhibit the growth of human leukemia HL-60 cells by G1 phase cell cycle arrest as a result of inhibited pRb phosphorylation.

Protective effects of dried flower extracts of Hibiscus sabdariffa L. against oxidative stress in rat primary hepatocytes.

Dried flower extracts of Hibiscus sabdariffa L., a local soft drink material and medical herb, was found to possess antioxidant activity in the present study. In the preliminary studies, antioxidant potential of three fractions of the ethanol crude extract (HS-C: chloroform-soluble fraction; HS-E: ethyl acetate soluble fraction; HS-R: residual fraction) obtained from the dried flowers of Hibiscus sabdariffa L. were evaluated by their capacity of quenching 1,1 -diphenyl-2-picrylhydrazyl (DPPH) free radical and inhibiting xanthine oxidase (XO) activity. HS-E showed the greatest capacity of scavenging free radical (EC50=0.017mg/ml), and HS-C showed the strongest inhibitory effect on XO activity (EC5o=0.742 mg/ml). Furthermore, antioxidant bioactivities of these crude extracts were investigated using a model of tert-butyl hydroperoxide (t-BHP)-induced oxidative damage in rat primary hepatocytes. All fractions were found to inhibit significantly the unscheduled DNA synthesis (UDS) induced by t-BHP at a concentration of 0.20 mg/ml. HS-C and HS-E also decreased the leakage of lactate dehydrogenase (LDH) and the formation of malondialdehyde (MDA) induced by t-BHP (1.5 mM) considerably at a concentration of 0.10 and 0.20 mg/ml in the rat primary hepatocyte cultures. These results indicated that the dried flower extracts (HS-C and HS-E) of H. sabdariffa L. protect rat hepatocytes from t-BHP-induced cytotoxicity and genotoxicity by different mechanisms.

Induction of apoptosis by Hibiscus protocatechuic acid in human leukemia cells via reduction of retinoblastoma (RB) phosphorylation and Bcl-2 expression.

Hibiscus protocatechuic acid (PCA), a phenolic compound isolated from the dried flower of Hibiscus sabdariffa L. (Malvaceae), demonstrated antioxidant and antitumor promotion effects in our previous study. In the present study, Hibiscus PCA was found to inhibit the survival of human promyelocytic leukemia HL-60 cells in a concentration- and time-dependent manner. The study revealed that HL-60 cells underwent internucleosomal DNA fragmentation and morphological changes characteristic of apoptosis after a 9-hr treatment with Hibiscus PCA (2 mM). Flow cytometric analysis of the DNA content of cells treated with PCA for 12 hr showed that the cells were distributed mainly in the hypodiploid phase (apoptotic peak, 46.7%), less in the G(1) (34.2%) and S phase (14.0%), and few in the G(2)/M phase (5.1%). Moreover, PCA treatment caused an increase in the level of hypophosphorylated retinoblastoma (RB; 180% of control at the 6-hr time point) and, on the contrary, a decline in hyperphosphorylated RB. A rapid loss of RB was observed when the treatment period was extended. Further studies showed that Hibiscus PCA application reduced Bcl-2 protein expression to 47%, and increased Bax protein expression to 181% after 1.5 hr as compared with time 0. Overexpression of Bcl-2 in HL-60 cells delayed the occurrence of Hibiscus PCA-induced apoptosis. These data suggest that Hibiscus PCA is an apoptosis inducer in human leukemia cells, and that RB phosphorylation and Bcl-2 protein may play a crucial role in the early stage.

Inhibitory effect of Hibiscus protocatechuic acid on tumor promotion in mouse skin

Hibiscus protocatechuic acid (PCA), a phenolic acid isolated from Hibiscus sabdariffa L., was evaluated for its ability to inhibit the 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced promotion in skin tumors of female CD-1 mice. Topical application of PCA (5, 10 or 20 micromol) 5 min prior to TPA (15 nmol) treatment twice weekly for 20 weeks to mice which were initiated with benzo[a]pyrene (B[a]P) inhibited the incidence of tumors in mice to 81.3, 62.5 and 56.3%, respectively, while all mice in the TPA-treated group developed tumors. The average number of tumors in mice pretreated with PCA was 2-4 and that of mice treated only with TPA was 6.6. The protection effects of PCA were also presented by its significant suppression on the TPA-induced hyperplasia in the skin and edema of mouse ears by 65 and 73% at doses of 10 and 20 micromol, respectively. When it was applied to the dorsal surface of CD-1 mice before TPA application, PCA (5, 10 or 20 micromol) inhibited the induction of epidermal ornithine decarboxylase (ODC) activity by 5 nmol TPA and myeloperoxidase (MPO) activity by 6.5 nmol TPA. The same doses of PCA also reduced the formation of hydrogen peroxide in the mouse skin to an inhibition of 61, 84 and 89%, respectively, when compared with that of the TPA-treated group. These results indicate that PCA possesses potential as a cancer chemopreventive agent against tumor promotion.

Crocetin protects against oxidative damage in rat primary hepatocytes.

Crocetin is a major component in the fruit of Gardenia jaminoides Ellis, a Chinese herbal medicine. Its protective action and mechanism against oxidative damage were investigated and mechanism against oxidative damage were investigated. Reactive oxygen species (ROS) were generated enzymatically in the xanthine-xanthine oxidase (X/XO 5 microM/0.01 u/ml) system and non-enzymatically in the paraquat (PQ 5 mM) system. Both systems increased leakage of lactate dehydrogenase (LDH) and alanine transaminase (ALT) in rat primary hepatocytes, but the hepatotoxicity was significantly suppressed on pretreatment with crocetin (10, 20 microM). Crocetin decreased formation of malondialdehyde (MDA) as an index of lipid peroxidation induced by ROS. The oxyradical generation by X/XO or PQ caused DNA damage evaluated with unscheduled DNA synthesis (UDS) in rat primary hepatocytes. The addition of crocetin decreased genotoxicity evaluated with UDS in both systems. The data showed that crocetin also inhibited the formation of superoxide anion in the X/XO system and bleached the free radical 1, 1-diphenyl-2- picrylhydrazyl (DPPH). The protective action of crocetin operated via quenching of the superoxide anion and/or free radical.

Hibiscus protocatechuic acid protects against oxidative damage induced by tert-butylhydroperoxide in rat primary hepatocytes

Hibiscus protocatechuic acid (PCA), a simple phenolic compound isolated from Hibiscus sabdariffa L., was studied for its protective effects against oxidative damage induced by tert-butylhydroperoxide (t-BHP) in a primary culture of rat hepatocytes. It had been reported that exposure of isolated hepatocytes to t-BHP results in leakage of lactate dehydrogenase (LDH) and alanine transaminase (ALT), peroxidation of cellular lipids, and depolarization of mitochondria. The present investigations showed that PCA at concentrations of 0.05 mg/ml and 0.10 mg/ml significantly decreased the leakage of LDH (P < 0.01) and ALT (P < 0.05 and P < 0.01) and the formation of malondialdehyde (MDA; P < 0.05 and P < 0.01) induced by 30-min treatment with t-BHP (1.5 mM) in primary cultured rat hepatocytes. PCA also attenuated t-BHP (0.10 mM) induced mitochondrial depolarization as determined by a retention test of rhodamine 123 and DNA repair synthesis as evidenced by unscheduled DNA synthesis (UDS). In addition, PCA exhibited an effective ability to quench 1,1-diphenyl-2-picrylhydrazyl radicals (DPPH). In conclusion, PCA demonstrated protective effects against cytotoxicity and genotoxicity of hepatocytes induced by t-BHP. One of mechanisms of PCAs protective effect may be associated with its property of scavenging free radicals.

Induction of apoptosis by esculetin in human leukemia cells

Esculetin, a coumarin compound, has been shown to exhibit antioxidant and anti-inflammatory effects. In the present study, esculetin was found to inhibit the survival of human promyelocytic leukemia HL-60 cells in a concentration-dependent and time-dependent manner. HL-60 cells underwent internucleosomal DNA fragmentation and morphological changes characteristic of apoptosis after a 24-h treatment with esculetin (100 microM). Flow cytometric analysis showed that the hypodiploid nuclei of HL-60 cells were increased to 40.93% after a 36-h treatment with esculetin (100 microM). Further investigation showed that esculetin induced the release of cytochrome c from mitochondria into cytosol in a time-dependent and concentration-dependent manner. Moreover, esculetin application reduced Bcl-2 protein expression to 58% after 9 h as compared with that time at 0. Cysteine protease 32 kDa proenzyme (CPP32), a caspase 3, was activated and its substrate, poly (adenosine diphosphate-ribose) polymerase, was cleaved after a 24-h treatment of HL-60 cells with esculetin. These data suggest that esculetin induces apoptosis in human leukemia cells by increasing cytosolic translocation of cytochrome c and activation of CPP32.

Protective effects of capillarisin on tert-butylhydroperoxide-induced oxidative damage in rat primary hepatocytes.

Abstract Capillarisin (Cap), a main constituent of Artemisia capillaris (Compositae), was studied for its antioxidant bioactivity. In the preliminary study, Cap expressed a antioxidant property by its capacity for quenching the free radicals of 1,1-diphenyl-2-picrylhydrazyl (DPPH). This antioxidant bioactivity of Cap was investigated further using a model of t-butylhydroperoxide (t-BHP)-induced cytotoxicity and genotoxicity in rat primary hepatocytes. Results presented here demonstrate that Cap, at concentrations of 0.01–1.00 mg/ml, significantly decreased the leakage of lactate dehydrogenase (LDH) and alanine aminotransferase (ALT) and the formation of malondialdehyde (MDA) induced by 30 min treatment of t-BHP (1.5 mM) in primary cultured rat hepatocytes. Cap also attenuated the t-BHP-induced diminution of glutathione (GSH) and high level of DNA repaired synthesis. These results lead to speculation that Cap presents inhibitory effects against t-BHP-caused cytotoxicity and genotoxicity in rat primary hepatocyte cultures at least via two distinct pathways, stabilizing the GSH system and quenching free radicals.

Antitumor progression potential of caffeic acid phenethyl ester involving p75NTR in C6 glioma cells

The previous data showed that caffeic acid phenethyl ester (CAPE), a component of propolis, possesses inducing cell cycle arrest and antiproliferation effect on C6 glioma cells in vitro and in vivo. In the present study, C6 glioma cells treated with CAPE resulted in morphological changes to an astrocytic phenotype and increased the expression of glial differentiation marker proteins including glial fibrillary acidic protein (GFAP) and S-100β. In addition, with scratch assay and Boyden chamber assay, CAPE exhibited inhibitory effects on the motility and invasion of C6 glioma cells. Furthermore, CAPE induced the expression of nerve growth factor (NGF) and p75 neurotrophin receptor (p75(NTR)), which were involved in neural cell differentiation. CAPE could also inhibit the activity of matrix metalloproteinases (MMPs) and induce the expression of RhoB, a tumor suppressor. To examine the involvement of p75(NTR) in the anti-invasive property of CAPE, Western blotting and Boyden Chamber assay were performed by addition of an anti-p75(NTR) antibody in C6 cells. The results showed that blocking p75(NTR) could decrease the CAPE-induced expression of RhoB and the inactivation of MMP-2, -9 as well as the anti-invasion effect in C6 glioma cells. Furthermore, CAPE suppressed IκB-α phosphorylation which was down stream of p75(NTR). Finally, the effect of CAPE on metastasis by lung colonization of the tumor cell in nude mice was also evaluated. It was found that the groups of nude mice injected with CAPE-pretreated cells could decrease both lung size and weight as compared to the positive control group which did not receive CAPE treatment. In addition, histological examination of the mouse lung sections showed that the CAPE-treated group inhibited the metastasis of C6 glioma cells. These data suggest CAPE possesses antitumor progression potential.