Effect of alanine replacement of l17 and f19 on the aggregation and neurotoxicity of arctic-type aß40.

Alzheimer's disease is the most common form of neurodegenerative disease. Beta-amyloid peptides (Aß) are responsible for neuronal death both in vitro and in vivo. Previously, L17 and F19 residues were identified as playing key roles in the stabilization of the Aß40 conformation and in the reduction of its neurotoxicity. In this study, the effects of L17A/F19A mutations on the neurotoxicity of Aß genetic mutant Arctic-type Aß40(E22G) were tested. The results showed that compared to Aß40(E22G), Aß40(L17A/F19A/E22G) reduced the rate of conformation conversion, aggregation, and cytotoxicity, suggesting that L17 and F19 are critical residues responsible for conformational changes which may trigger the neurotoxic cascade of Aß. Aß40(L17A/F19A/E22G) also had decreased damage due to reactive oxygen species. The results are consistent with the discordant helix hypothesis, and confirm that residues 17-25 are in the discordant helix region. Compared to Aß40(L17A/F19A), reduction in aggregation of Aß40(L17A/F19A/E22G) was less significantly decreased. This observation provides an explanation based on the discordant helix hypothesis that the mutation of E22 to G22 of Aß40(E22G) alters the propensity of the discordant helix. Arctic-type Aß40(E22G) aggregates more severely than wild-type Aß40, with a consequential increase in toxicity.

Baicalein protects against retinal ischemia by antioxidation, antiapoptosis, downregulation of HIF-1α, VEGF, and MMP-9 and upregulation of HO-1.

PURPOSE: Retinal ischemia-associated ocular disorders are vision threatening. This study examined whether the flavonoid baicalein is able to protect against retinal ischemia/reperfusion. METHODS: Using rats, the intraocular pressure was raised to 120 mmHg for 60 min to induce retinal ischemia. In vitro, an ischemic-like insult, namely oxidative stress, was established by incubating dissociated retinal cells with 100 µM ascorbate and 5 µM FeSO4 (iron) for 1 h. The rats or the dissociated cells had been pretreated with baicalein (in vivo: 0.05 or 0.5 nmol; in vitro: 100 µM), vehicle (1% ethanol), or trolox (in vivo: 5 nmol; in vitro: 100 µM or 1 mM). The effects of these treatments on the retina or the retinal cells were evaluated by electrophysiology, immunohistochemistry, terminal deoxynucleotidyl-transferase-mediated dUTP nick end-labeling (TUNEL) staining, Western blotting, or in vitro dichlorofluorescein assay. In addition, real-time-polymerase chain reaction was used to assess the retinal expression of hypoxia-inducible factor-1α (HIF-1α), matrix metalloproteinase-9 (MMP-9), vascular endothelium growth factor (VEGF), and heme oxygenase-1 (HO-1). RESULTS: The retinal changes after ischemia included a decrease in the electroretinogram b-wave amplitude, a loss of choline acetyltransferase immunolabeling amacrine cell bodies/neuronal processes, an increase in vimentin immunoreactivity, which is a marker for Müller cells, an increase in apoptotic cells in the retinal ganglion cell layer linked to a decrease in the Bcl-2 protein, and changes in the mRNA levels of HIF-1α, VEGF, MMP-9, and HO-1. Of clinical importance, the ischemic detrimental effects were concentration dependently and/or significantly (0.05 nmol and/or 0.5 nmol) altered when baicalein was applied 15 min before retinal ischemia. Most of all, 0.5 nmol baicalein significantly reduced the upregulation of MMP-9; in contrast, 5 nmol trolox only had a weak attenuating effect. In dissociated retinal cells subjected to ascorbate/iron, there was an increase in the levels of reactive oxygen species, which had been significantly attenuated by 100 µM baicalein and trolox (100 µM or 1 mM; a stronger antioxidative effect at 1 mM). CONCLUSIONS: Baicalein would seem to protect against retinal ischemia via antioxidation, antiapoptosis, upregulation of HO-1, and downregulation of HIF-1α, VEGF, and MMP-9. The antioxidative effect of baicalein would appear to play a minor role in downregulation of MMP-9.

Therapeutic effects and mechanisms of action of mannitol during H2O2-induced oxidative stress in human retinal pigment epithelium cells.

BACKGROUND: Age-related macular degeneration (AMD) is a leading cause of blindness in the elderly. At a later stage, neovascular or exudative AMD can lead to severe central vision loss that is related to aging-associated cumulative oxidative stress of the human retinal pigment epithelium (hRPE) and choroid capillary. Early prevention with antioxidants is mandatory. The aim of this study was to determine whether and how mannitol can act as an antioxidant. METHODS: The methods used included measurements of cell viability, oxygen free radical (OFR) levels, lipid peroxide (LP) levels, and OFR-related enzyme protein levels. RESULTS: H(2)O(2) dose-dependently reduced the cell viability of hRPE cells. This negative effect was significantly counteracted by pretreatment with mannitol (1 mM). H(2)O(2) significantly stimulated the formation of OFR and LP. These increases were dose-dependently and significantly blunted by mannitol. Furthermore, treatment with H(2)O(2) was associated with a reduction in the level of catalase, but not of manganese superoxide dismutase (MnSOD). In contrast, it was shown that mannitol protected hRPE cells against the H(2)O(2)-induced oxidative stress by increasing the level of catalase, but not the level of MnSOD. CONCLUSION: This study supports an antioxidative role for mannitol that acts through up-regulating the level of catalase, which is decreased by H(2)O(2).

Hepatoprotective effect of shidagonglao on acute liver injury induced by carbon tetrachloride.

This study investigates the hepatoprotective activity of ethanol extract from Shidagonglao roots (SDGL(EtOH)). The hepatoprotective effect of SDGL(EtOH) (20, 100 and 500 mg/kg) was analyzed on carbon tetrachloride (CCl(4))-induced acute liver injury. Rats pretreated orally with SDGL(EtOH) (100 and 500 mg/kg) and silymarin (200 mg/kg) for 3 consecutive days prior to the administration of a single dose of 50% CCl(4) (0.10 ml/100 g of bw, ip) significantly prevented the increases in the activities of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in CCl(4)-treated rats. Histological analysis also showed that SDGL(EtOH) (100 and 500 mg/kg) and silymarin reduced the incidence of liver lesions including vacuole formation, neutrophil infiltration and necrosis of hepatocytes induced by CCl(4) in rats. Moreover, the SDGL(EtOH) (100 and 500 mg/kg) increased the activities of anti-oxidative enzymes, superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione reductase (GRd) and decreased malondialdehyde (MDA) level in liver, as compared to those in the CCl(4)-treated group. Furthermore, SDGL(EtOH) (100 and 500 mg/kg) and silymarin attenuated the increased levels of tumor necrosis factor-alpha (TNF-alpha) in serum and nitric oxide (NO) in liver as compared to the CCl(4)-treated group. The hepatoprotective mechanisms of SDGL(EtOH) are likely related to inhibition of TNF-alpha, MDA and NO productions via increasing the activities of antioxidant enzymes (SOD, GPx and GRd). These experimental results suggest that SDGL(EtOH) can attenuate CCl(4)-induced acute liver injury in rats.

Analgesic and anti-inflammatory activities of ethanol root extract of Mahonia oiwakensis in mice.

AIMS OF THE STUDY: This study investigated the anti-inflammatory and analgesic activities, and protoberberine alkaloid contents of ethanol extract of MO roots (MOR(EtOH)). MATERIALS AND METHODS: The analgesic activity of MOR(EtOH) was determined using acetic acid-induced writhing response and formalin test. The anti-inflammatory activity of MOR(EtOH) was determined using the lambda-carrageenan-induced paw oedema model. The protoberberine alkaloid contents of MOR(EtOH) were identified by high-performance liquid chromatography (HPLC). RESULTS: MOR(EtOH) (100 and 500 mg/kg) decreased the acetic acid-induced writhing responses and licking times of the second phase in the formalin test. Moreover, carrageenan-induced paw oedema was significantly reduced in a dose-dependent manner by administering MOR(EtOH) (100 and 500 mg/kg) at 3, 4, and 5h after the carrageenan injection. The serum levels of tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) of MOR(EtOH)-treated mice were significantly reduced compared with those in the serum of animals administered carrageenan. Notably, MOR(EtOH) attenuated the expression of cyclo-oxygenase 2 (COX-2) and inducible nitric oxide synthase (iNOS) and neutrophil infiltration in paw tissues injected with carrageenan. The anti-inflammatory mechanisms of MOR(EtOH) appear to be related to the inhibition of neutrophil infiltration, iNOS and COX-2 protein expression, NO release, and the decreasing TNF-alpha level in serum. The analytical results showed that the contents of berberine, palmatine and jatrorrhizine were 191.45 mg/g extract, 100.15 mg/g extract and 66.45 mg/g extract, respectively. CONCLUSION: These experimental results suggest that MOR(EtOH) produced both analgesic and anti-inflammatory effects in mice and may be a candidate for the development of pharmacological agents used in the treatment of inflammatory disorders.

Anticancer effects of low-dose 10-hydroxycamptothecin in human colon cancer.

10-Hydroxycamptothecin (10-HCPT), an indole alkaloid isolated from a Chinese tree, Camptotheca acuminate, inhibits the activity of topoisomerase I and has a broad spectrum of anticancer activity in vitro and in vivo. However, its use has been limited due to its water-insolubility and toxicity with i.v. administration. The purpose of this study was to investigate the efficacy, toxicity and proper dosage of 10-HCPT as a single agent by oral administration in the treatment of human colon cancer. 10-HCPT significantly repressed the proliferation of Colo 205 cells at a relatively low concentration (5-20 nM). Flow cytometry analysis and western blot and apoptosis assays demonstrated that low-dose 10-HCPT arrested Colo 205 cells in the G2 phase of the cell cycle and triggered apoptosis through a caspase-3-dependent pathway. Moreover, following oral administration at doses of 2.5-7.5 mg/kg/2 days, significant suppression of tumor growth by 10-HCPT was observed in mouse xenografts. No acute toxicity was observed after an oral challenge of 10-HCPT in BALB/c-nude mice every 2 days. The results of this study suggest that a relatively low dose of 10-HCPT (p.o.) is able to inhibit the growth of colon cancer, facilitating the development of a new protocol of human trials with this anticancer drug.

Oxidative metabolism of the alkaloid rutaecarpine by human cytochrome P450.

Rutaecarpine is the main active alkaloid of the herbal medicine, Evodia rutaecarpa. To identify the major human cytochrome P450 (P450) participating in rutaecarpine oxidative metabolism, human liver microsomes and bacteria-expressed recombinant human P450 were studied. In liver microsomes, rutaecarpine was oxidized to 10-, 11-, 12-, and 3-hydroxyrutaecarpine. Microsomal 10- and 3-hydroxylation activities were strongly inhibited by ketoconazole. The 11- and 12-hydroxylation activities were inhibited by alpha-naphthoflavone, quinidine, and ketoconazole. These results indicated that multiple hepatic P450s including CYP1A2, CYP2D6, and CYP3A4 participate in rutaecarpine hydroxylations. Among recombinant P450s, CYP1A1 had the highest rutaecarpine hydroxylation activity. Decreased metabolite formation at high substrate concentration indicated that there was substrate inhibition of CYP1A1- and CYP1A2-catalyzed hydroxylations. CYP1A1-catalyzed rutaecarpine hydroxylations had V(max) values of 1,388 to approximately 1,893 pmol/min/nmol P450, K(m) values of 4.1 to approximately 9.5 microM, and K(i) values of 45 to approximately 103 microM. These results indicated that more than one molecule of rutaecarpine is accessible to the CYP1A active site. The major metabolite 10-hydroxyrutaecarpine decreased CYP1A1, CYP1A2, and CYP1B1 activities with respective IC(50) values of 2.56 +/- 0.04, 2.57 +/- 0.11, and 0.09 +/- 0.01 microM, suggesting that product inhibition might occur during rutaecarpine hydroxylation. The metabolite profile and kinetic properties of rutaecarpine hydroxylation by human P450s provide important information relevant to the clinical application of rutaecarpine and E. rutaecarpa.

Identification of the microsomal oxidation metabolites of rutaecarpine, a main active alkaloid of the medicinal herb Evodia rutaecarpa.

Rutaecarpine is a quinazolinocarboline alkaloid of the medicinal herb Evodia rutaecarpa and shows a variety of pharmacological effects. Four oxidation metabolites of rutaecarpine were prepared from 3-methylcholanthrene-treated rat liver microsomes. These metabolites had an [M + H]+ ion at m/z 304. The structures of metabolites were identified by comparison of their liquid chromatograms and mass, absorbance, and 1H NMR spectra with those of synthetic standards. Rutaecarpine was metabolized by microsomal enzymes to form 3-, 10-, 11-, and 12-hydroxyrutaecarpine. The formation of 10-hydroxyrutaecarpine was highly induced by a cytochrome P450 1A inducer, 3-methylcholanthrene.

Cytotoxicity of phenolic acid phenethyl esters on oral cancer cells.

Many phenolic acid phenethyl esters possess diverse biological effects including anti-cancer activity. A series of 14 derivatives were synthesized for the evaluation of their cytotoxic effect on oral cancer cells. These derivatives were tested by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric and trypan blue dye exclusion assay on the growth of oral squamous cell carcinoma (SAS), oral epidermoid carcinoma-Meng 1 (OEC-M1), and normal human oral fibroblast (NHOF) cells, respectively. Caffeic acid phenethyl esters, 3a (CAPE), and 3b, 3c, and 3d showed cytotoxic effects on the SAS and OEC-M1 cell lines, but not the NHOF cell line at a 5-100 microM dose range. Flow cytometric analysis showed that 3c caused OEC-M1 cell arrest at G2/M phase. Such differential effects on representative cancer and normal cells suggested these compounds might be useful in oral cancer chemotherapy.

Effects of phenolic acid esters and amides on stimulus-induced reactive oxygen species production in human neutrophils.

BACKGROUND: Phenolic acids and their derivatives are widely distributed in plants. A series of phenolic acid esters and amides have been synthesized. METHODS: We determined the effects of phenolic acid derivatives on antiinflammatory activity against phorbol 12-myristate 13-acetate (PMA) and N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced production of superoxide anion, an inflammatory mediator produced by neutrophils. RESULTS: When the cells were preincubated with phenolic acids and their derivatives, the superoxide generation induced by fMLP (1.0 micromol/l) and PMA (0.16 micromol/l) was inhibited to various degrees with compounds 1, 2 and 4 significantly suppressing such generation in a concentration-dependent manner. CONCLUSION: Phenolic acid derivatives may exert their antiinflammatory action through inhibiting superoxide generation.

Identification of the main human cytochrome P450 enzymes involved in safrole 1'-hydroxylation.

Safrole is a natural plant constituent, found in sassafras oil and certain other essential oils. The carcinogenicity of safrole is mediated through 1'-hydroxysafrole formation, followed by sulfonation to an unstable sulfate that reacts to form DNA adducts. To identify the main cytochrome P450 (P450) involved in human hepatic safrole 1'-hydroxylation (SOH), we determined the SOH activities of human liver microsomes and Escherichia coli membranes expressing bicistronic human P450s. Human liver (n = 18) microsomal SOH activities were in the range of 3.5-16.9 nmol/min/mg protein with a mean value of 8.7 +/- 0.7 nmol/min/mg protein. In human liver (n = 3) microsomes, the mean K(m) and V(max) values of SOH were 5.7 +/- 1.2 mM and 0.14 +/- 0.03 micromol/min/nmol P450, respectively. The mean intrinsic clearance (V(max)/K(m)) was 25.3 +/- 2.3 microL/min/nmol P450. SOH was sensitive to the inhibition by a CYP2C9 inhibitor, sulfaphenazole, and CYP2E1 inhibitors, 4-methylpyrazole and diethyldithiocarbamate. The liver microsomal SOH activity showed significant correlations with tolbutamide hydroxylation (r = 0.569) and chlorzoxazone hydroxylation (r = 0.770) activities, which were the model reactions catalyzed by CYP2C9 and CYP2E1, respectively. Human CYP2C9 and CYP2E1 showed SOH activities at least 2-fold higher than the other P450s. CYP2E1 showed an intrinsic clearance 3-fold greater than CYP2C9. These results demonstrated that CYP2C9 and CYP2E1 were the main P450s involved in human hepatic SOH.

Evaluation of the anti-inflammatory and cytotoxic effects of anthraquinones and anthracenes derivatives in human leucocytes.

A variety of anthracene- and anthraquinone-related derivatives, modified from three types of lead structures, including 9-acyloxy 1,5-dichloroanthracene (type I), 1,5-bisacyloxy-anthraquinones with O-linked substituents (type II) and 1,5-bisacyloxy-anthraquinones with S-linked substituents (type III), were synthesized and evaluated by an in-vitro bioassay for their anti-inflammatory and cytotoxic effects in human leucocytes. Among these derivatives, type I compounds displayed potent anti-inflammatory activity against phorbol-12-myristate-13-acetate (PMA)-induced superoxide anion production, a bio-marker of inflammatory mediator production by neutrophils, with 50% inhibition (IC50) concentrations (microM) for compounds 1f, 1g, 1h and 1m being 13.8 +/- 3.0, 6.3 +/- 4.1, 33.2 +/- 1.3 and 33.9 +/- 5.7, respectively. Type II and type III derivatives (i. e., 1,5-bisacyloxy anthraquinone-related compounds) and the reference compound, emodin, exhibited relatively minor (20-40%) inhibitory effect against superoxide production by neutrophils. Furthermore, none of these compounds showed a significant cytotoxic effect in human neutrophils. In conclusion, these results suggest that compounds modified from 9-acyloxy 1,5-dichloroanthracence (type I) are more powerful than the other two types as anti-inflammatory drugs. This is the first demonstration that derivatives modified from anthracenes or anthraquinones possess anti-inflammatory activity with no significant cytotoxicity in human neutrophils.

Small-molecule anthracene-induced cytotoxicity and induction of apoptosis through generation of reactive oxygen species.

A series of anthracene derivatives have been synthesized, and their potential individual cytotoxicity was evaluated using Jurkat T cells and peripheral blood mononuclear cells (PBMCs) in vitro. These compounds, except for 2l, showed less cytotoxicity in PBMCs than mitoxantrone. We also analyzed the antiproliferative activity of these derivatives using the annexin V/propidium iodide assay. These synthetic compounds induced apoptosis, thus leading to antitumor effects. Compounds 2b, 2e, 2f, 2g, 2h, 2i, 2j, and mitoxantrone produced dose-dependent cytotoxicity, while the antiproliferative activity of the anthracene pharmacophore was retained in Jurkat T cells base on the detection of DNA degradation and membrane unpacking. These clearly indicate a correlation between cytotoxicity and antitumor activity. Unlike mitoxantrone, cytotoxic properties were observed, as documented by the reactivity of these novel compounds against Jurkat T cells and PBMCs as normal cells, respectively. Various concentrations of 2b, 2e, 2f, 2g, 2h, 2i, and 2j preparations also inhibited Jurkat T cell proliferation and induced apoptosis of Jurkat T cells, potentially confirmed through the detection of DNA degradation and membrane unpacking. In the present report we also investigated the antiinflammatory activity against phorbol-12-myristate-13-acetate induced superoxide anion production, a marker for an inflammatory mediator produced by neutrophils, with IC(50) (microM) values of 2b, 2h, 2l, and 2o of 4.28+/-0.89, 3.31+/-0.88, 4.38+/-0.25, and 5.45+/-1.78, respectively. These results suggest that, in addition to the specific chromosomal aberrations and cell death, elevated apoptosis could also be a marker for exposure to anthracene derivatives.

Analysis of synthetic anti-diabetic drugs in adulterated traditional Chinese medicines by high-performance capillary electrophoresis.

Four synthetic anti-diabetic drugs, acetohexamide (ACE), chlorpropamide (CHL), glibenclamide (GLI) and tolbutamide (TOL), which can be found as adulterants in traditional Chinese medicines (TCMs) were assayed simultaneously using high-performance capillary electrophoresis (HPCE) in 4 min with UV detection at 200 nm. The electrolyte was a buffer solution containing 100 mM phosphate buffer (NaH2PO4/Na2B4O7, pH 7.5). Applied voltage was 15.0 kV and temperature was 30 degrees C. 2-(4-Hydroxyphenyl) ethyl ammonium chloride (HEA) was used as an internal standard. The effects of buffer concentration, pH and supplied voltage on separation were investigated. The relative standard deviations (R.S.D.) of these anti-diabetic drugs for intra-day and inter-day analyses were 0.23-4.27 and 1.23-6.33%, respectively. The recoveries of the synthetic drug adulterants in traditional Chinese medicinal formula ranged from 81.3 to 105.5%. GLI was found and determined in a real sample of TCM.

Determination of matrine and oxymatrine in Sophora subprostata by CE.

Shan-dou-gen is the dried roots of Sophora subprostata (Leguminosae) and a commonly used Chinese herbal drug in Taiwan. It possesses antipyretic, anti-inflammatory, analgesic effects and is used to treat sore throat and acute pharyngolaryngeal infections. To evaluate the quality of S. subprostata, a simple, rapid and accurate high-performance capillary electrophoresis (HPCE) method was developed for the assay of two alkaloids: matrine and oxymatrine. The electrolyte was a buffer solution containing 75% 130 mM phosphate buffer (NaH2PO4/H3PO4, pH 3.5) and 25% acetonitrile. Applied voltage was 10 kV and temperature was 30 degrees C. 2-(4-Hydroxyphenyl)ethylammonium chloride was used as an internal standard and detector set at 200 nm. The contents of matrine and oxymatrine of S. subprostata in several different samples of crude drugs and commercial concentrated preparation have also been determined.

Preventive effect of the Taiwan folk medicine Ixeris laevigata var. oldhami on alpha-naphthyl-isothiocyanate and carbon tetrachloride-induced acute liver injury in rats.

The hepatoprotective effects of Ixeris laevigata Sch-Bip. var. oldhami Kitam. (IL) were studied on cholestatic hepatitis induced by alpha-naphthylisothiocyanate (ANIT, 100 mg/10 mL/kg, in olive oil, i.p.) and acute hepatitis induced by carbon tetrachloride (20% CCl(4)/olive oil, 1.5 mL/kg, i.p.) in rats. Hepatoprotective activity was monitored by estimating the serum transaminases levels and the histopathological changes in the livers of experimental rats. The pretreatment of animals with IL, extract (0.3-2.0 g/kg orally) significantly inhibited the acute elevation of serum transaminases, as well as the hepatotoxin-induced histopathological changes in the livers of the experimental rats.

Synthesis and vasorelaxant activity of 4-(cyclic amido)-2H-naphtho[1,2-b]pyrans.

A series of 4-(cyclic amido)-2H-naphtho[1,2-b]pyrans related to cromakalim (1) has been prepared and their vasorelaxant activities on isolated rat thoracic aorta precontracted with phenylephrine have been evaluated. The relaxant mechanism of 3a was found not through ATP-sensitive K(+) channels as cromakalim, but through opening voltage-sensitive K(+) channels.