Rosehip inhibits xanthine oxidase activity and reduces serum urate levels in a mouse model of hyperuricemia.

Rosehip, the fruit of Rosa canina L., has traditionally been used to treat urate metabolism disorders; however, its effects on such disorders have not been characterized in detail. Therefore, the present study investigated the effects of hot water, ethanol and ethyl acetate extracts of rosehip on xanthine oxidase (XO) activity in vitro. In addition, the serum urate lowering effects of the rosehip hot water extract in a mouse model of hyperuricemia (male ddY mice, which were intraperitoneally injected with potassium oxonate) were investigated. Furthermore, the influence of rosehip hot water extract on CYP3A4 activity, which is the most important drug-metabolizing enzyme from a herb-drug interaction perspective, was investigated. Rosehip extracts of hot water, ethanol and ethyl acetate inhibited XO activity [half maximal inhibitory concentration (IC50) values: 259.6±50.6, 242.5±46.2 and 1,462.8±544.2 µg/ml, respectively]. Furthermore, the administration of 1X rosehip hot water extract significantly reduced the levels of serum urate at 8 h, which was similar when compared with the administration of 1 mg/kg allopurinol. Rosehip hot water extract only marginally affected CYP3A4 activity (IC50 value, >1 mg/ml). These findings indicate that rosehip hot water extract may present as a functional food for individuals with a high urate level, and as a therapeutic reagent for hyperuricemic patients.

Effects of tomato juice on the pharmacokinetics of CYP3A4-substrate drugs.

We previously demonstrated that tomato juice (TJ) contains potent mechanism-based inhibitor(s) of CYP3A4. In this study, we investigated the effects of TJ and grapefruit juice (GFJ) on the pharmacokinetics of the CYP3A4-substrate drugs, nifedipine (NFP) and midazolam (MDZ), in male Wistar rats. Oral administration of GFJ 90 min before the intraduodenal administration of NFP or MDZ increased the area under the concentration-time curves (AUCs) of NFP and MDZ by 32.4% and 89.4%, respectively. TJ increased MDZ blood concentrations and AUC after intraduodenal MDZ administration; however, it had no effect on NFP. When MDZ and NFP were intravenously administered, GFJ significantly increased the AUC of MDZ, but only slightly increased that of NFP. In contrast, TJ only slightly increased the AUC of MDZ. These results suggest that, similar to GFJ, TJ influences the pharmacokinetics of CYP3A4-substrate drugs; however, it may be a drug-dependent partial effect.

Efficient utilization of licorice root by alkaline extraction.

Compared to studies of water extracts of plants, those utilising alkaline extracts are limited. Both water and alkaline extracts from licorice root were compared regarding their biological activities. Licorice root was successively extracted first with water or alkaline solution (pH 9 or 12), and the alkaline (pH 12.0) extract was further separated into 50% ethanol-soluble and -insoluble fractions. Viable cell number was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. Antibacterial activity against Porphyromonas gingivalis 381 was determined by turbidity assay. Cytochrome P-450 (CYP)3A4 activity was measured by ß-hydroxylation of testosterone using human recombinant CYP3A4. Radical intensity of superoxide and hydroxyl radicals was determined by electron spin resonance spectroscopy. Alkaline extraction yielded slightly higher amounts of dried materials compared to water extraction. Alkaline extract showed higher anti-HIV and antibacterial activities, and similar magnitudes of CYP3A4 inhibitory and superoxide and hydroxyl radical-scavenging activities, compared to water extract. When alkaline extract was fractionated by 50% ethanol, anti-HIV activity was recovered from the insoluble fraction representing approximately 3% of the alkaline extract, whereas antibacterial activity was concentrated in the soluble fraction rich in glycyrrhizid acid, flavanones and chalcones. All extracts and sub-fractions led to bimodal hormetic dose-response (maximum hormetic response=238%) on the bacterial growth. The present study demonstrated the superiority of alkaline extraction over water extraction for preparing anti-HIV and antibacterial agents at higher yield from licorice root.

Signal transduction mechanism for potentiation by α1- and ß2-adrenoceptor agonists of L-ascorbic acid-induced DNA synthesis and proliferation in primary cultures of adult rat hepatocytes.

We investigated the effects of α- and ß-adrenoceptor agonists on L-ascorbic acid-induced hepatocyte DNA synthesis and proliferation in primary cultures of adult rat hepatocytes. The results showed that phenylephrine (10(-6) M) and metaproterenol (10(-6) M) alone did not induce hepatocyte DNA synthesis and proliferation. However, when combined with L-ascorbic acid (10(-6) M), these adrenoceptor agonists potentiated the hepatocyte DNA synthesis and proliferation induced by L-ascorbic acid. Then intracellular signal transduction mechanisms for the effects of phenylephrine and metaproterenol on L-ascorbic acid-induced hepatocyte mitogenesis were examined. Western blot analysis showed that phenylephrine and metaproterenol did not potentiate L-ascorbic acid-induced insulin-like growth factor I receptor tyrosine kinase phosphorylation. In contrast, they both significantly potentiated L-ascorbic acid-induced extracellular-signal regulated kinase-2 (ERK2) phosphorylation within 5 min. Moreover, cell-permeable second messenger analogs phorbol ester (10(-7) M) and 8-bromo cAMP (10(-7) M) mimicked the effects of phenylephrine and metaproterenol on L-ascorbic acid-induced ERK2 phosphorylation. The effects of these adrenoceptor agents were specifically antagonized by GF109203X and H-89, respectively. These results indicate that activation of ERK2 via protein kinas C and protein kinase A represents a mechanism for potentiation of L-ascorbic acid-induced hepatocyte DNA synthesis and proliferation in primary cultures of adult rat hepatocytes.

Biological activity of SE-10, granulated powder of Sasa senanensis Rehder leaf extract.

BACKGROUND: We have previously reported that alkaline extract of Sasa senanensis leaves (SE) showed potent anti-HIV, anti-UV and radical scavenging activity. In the present study, we investigated the biological activities of SE-10, a granulated powder of SE supplemented with lactose, lactitol, trehalose and tea extract. MATERIALS AND METHODS: Cell viability of mock-infected, HIV-infected, and UV-irradiated cells was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. Scavenging activity of superoxide anion and hydroxyl radicals was determined by electron-spin resonance spectroscopy. Cytochrome P-450 (CYP)3A4 activity was measured by ß-hydroxylation of testosterone in human recombinant CYP3A4. RESULTS: SE-10 had slightly higher anti-HIV and anti-UV activities, but slightly lower radical-scavenging and CYP3A4-inhibitory activities, as compared with SE. CONCLUSION: The present study demonstrates that the biological activities of SE were well preserved during the manufacturing process of SE-10.

Mechanism-based inhibition of recombinant human cytochrome P450 3A4 by tomato juice extract.

This study investigates whether tomato juice can inhibit cytochrome P450 (CYP) 3A4-mediated drug metabolism. Three commercially available, additive-free tomato juices, along with homogenized fresh tomato, were analyzed for their ability to inhibit testosterone 6ß-hydroxylation activity using human recombinant CYP3A4. Results were compared to that of grapefruit juice. Ethyl acetate extracts of the tomato juices moderately reduced residual activity of CYP3A4 testosterone 6ß-hydroxylation activity by 19.3-26.2% with 0-min preincubation. Residual activity was strongly reduced by 69.9-83.5% at 20-min preincubation, a reduction similar to that of grapefruit juice extract, known to contain constituents of mechanism-based inhibitors. One juice extract (tomato juice C) showed irreversible dose- and preincubation time-dependent and partial nicotinamide adenine dinucleotide phosphate (NADPH)-dependent inhibition of CYP3A4 activity. Furthermore, we examined whether the CYP3A4 inhibitory effect of tomato juice was substrate dependent by examining midazolam 1'-hydroxylation activity and nifedipine oxidation activity, in addition to testosterone 6ß-hydroxylation activity. Tomato juice showed a potent inhibitory effect on nifedipine oxidation activity, which was comparable to that on testosterone 6ß-hydroxylation activity; however, it showed a weak inhibitory effect on midazolam 1'-hydroxylation activity. We conclude that tomato juice contains one or more mechanism-based and competitive inhibitor(s) of CYP3A4. Additionally, significant CYP3A4 inhibitory activity did not result from lycopene, a major compound in tomato. Although the active compound was uncertain, a strong CYP3A4 inhibitory activity was observed in other solanaceous plants, i.e., potato, eggplant, sweet pepper, and capsicum. Therefore, responsible compounds in tomato are likely commonly shared among solanaceous vegetables.

Comparative study of biological activity of three commercial products of Sasa senanensis Rehder leaf extract.

BACKGROUND: We have previously reported that alkaline extract of Sasa senanensis leaves (SE) has several biological activities characteristic of lignin-carbohydrate complex (LCC). In the present study, we compared the biological activity of three commercially available products of SE (products A, B and C). MATERIALS AND METHODS: Cell viability of mock-infected, HIV-infected, UV-irradiated cells was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. Radical intensity was determined by electron spin resonance spectroscopy. Cytochrome P-450 (CYP)3A4 activity was measured by ß-hydroxylation of testosterone in human recombinant CYP3A4. RESULTS: Product A is a pure SE that contains Fe(II)-chlorophyllin, whereas products B and C contain Cu(II)-chlorophyllin and less LCC. Product C is supplemented with ginseng and pine (Pinus densiflora) leaf extracts. Product A exhibited 5-fold higher anti-HIV, 4-fold higher anti-UV, 5-fold higher hydroxyl radical-scavenging, and 3-fold lower CYP3A4 inhibitory activities as compared to those of product B, and 5-fold higher, 1.5-fold higher, comparable, and 7-fold lower activities, respectively, as compared to those of product C. CONCLUSION: The present study demonstrates for the first time the superiority of product A over products B and C, suggesting the beneficial role of LCC and Fe(II)-chlorophyllin.

Differential induction of stearoyl-CoA desaturase 1 and 2 genes by fibrates in the liver of rats.

The administration of fibrates (fenofibrate, bezafibrate and clofibric acid) to rats induced stearoyl-CoA desaturase (SCD) in the liver, and increased relative expression of mRNAs encoding SCD1 and SCD2 in dose- and time-dependent manners. The magnitudes of the increases in SCD2 mRNA level caused by fenofibrate and clofibric acid were much higher than those of SCD1 at relatively higher doses of the fibrates, and a relatively long time (7 or 14 d) was required for significant induction of SCD2 mRNA expression compared with that of SCD1. Although the absolute number of transcripts for SCD2 was 1,800 times lower than that of SCD1 in the control liver, it was strikingly increased by fibrates. These results suggest that differential regulations operate for the gene expression between SCD1 and SCD2, and that the physiological significance of SCD2 is distinct from that of SCD1 in the liver.

Clofibric acid increases the formation of oleic acid in endoplasmic reticulum of the liver of rats.

The effects of 2-(4-chlorophenoxy)-2-methylpropionic acid (clofibric acid) on the formation of oleic acid (18:1) from stearic acid (18:0) and utilization of the 18:1 formed for phosphatidylcholine (PC) formation in endoplasmic reticulum in the liver of rats were studied in vivo. [¹4C]18:0 was intravenously injected into control Wistar male rats and rats that had been fed on a diet containing 0.5% (w/w) clofibric acid for 7 days; and the distribution of radiolabeled fatty acids among subcellular organelles, microsomes, peroxisomes, and mitochondria, was estimated on the basis of correction utilizing the yields from homogenates of marker enzymes for these organelles. The radioactivity was mostly localized in microsomes and the radiolabeled fatty acids present in microsomes were significantly increased by the treatment of rats with clofibric acid. The formation of radiolabeled 18:1 in microsomes markedly increased and incorporations of the formed [¹4C]18:1 into PC and phosphatidylethanolamine in microsomes were augmented in response to clofibric acid. The [¹4C]18:1 incorporated into PC was mostly located at the C-2 position, but not the C-1 position, of PC, and the radioactivity in 18:1 at the C-2 position of PC was strikingly increased by clofibric acid. These results obtained from the in vivo experiments directly link the findings that clofibric acid treatment induces microsomal stearoyl-CoA desaturase and 1-acylglycerophosphocholine acyltransferase in the liver and the findings that the treatment with the drug elevated absolute mass and mass proportion of 18:1 at the C-2 position, but not the C-1 position, of PC in the liver together.

Effects of perfluorinated fatty acids with different carbon chain length on fatty acid profiles of hepatic lipids in mice.

Alterations by perfluorinated fatty acids (PFCAs) with a chain length of 6-9 carbons in the fatty acid profile of hepatic lipids of mice were investigated. The characteristic changes caused by all the PFCAs examined were increases in the contents and proportions of oleic acid (18 : 1), palmitoleic acid (16 : 1) and 8,11,14-eicosatrienoic acid (20 : 3) in hepatic lipids. Hepatic contents of palmitic acid were also increased by the treatments with the PFCAs. These effects were almost dependent on the hepatic concentrations of PFCA molecules regardless of their carbon chain length. Perfluorooctanoic acid elevated the expressions of mRNA encoding acetyl-CoA carboxylase, fatty acid synthase, malic enzyme, stearoyl-CoA desaturase (SCD) (SCD1 and 2), chain elongase (ELOVL5), Δ6 desaturase (Fads2), 1-acylglycerophosphocholine acyltransferase (LPCAT) (LPCAT3). The four PFCAs examined induced microsomal SCD and LPCAT in hepatic concentration-dependent manners regardless of carbon chain length. One linear regression line was confirmed between LPCAT activity and hepatic concentration of PFCA at wide range of the concentration, whereas the induction of SCD was saturable at relatively low concentration of PFCAs. These results suggest (i) that PFCAs with a chain length of 6-9 carbons change the fatty acid profile of hepatic lipids by increasing contents and proportions of 16 : 1, 18 : 1 and 20 : 3, (ii) that these alterations in fatty acid profile are caused by up-regulation of SCD, de novo fatty acid synthesis, chain elongase and Δ6 desaturase and (iii) that the mechanism underlying SCD induction is, in part, mediated through peroxisome proliferator-activated receptor α.

Peony root extract upregulates transthyretin and phosphoglycerate mutase in mouse cobalt focus seizure.

Cobalt focus is a seizure focus model in which cerebral neurons exhibit long-lasting severe spike discharges, followed by neuronal death. However, the neuronal death is prevented when peony root extract (PR) is administered prior to cobalt application. We tested the hypothesis that PR modulates the expression of neuroprotective proteins in the cerebrum of mouse cobalt focus by proteomic analysis using two-dimensional polyacrylamide gel electrophoresis and mass spectrometry to screen for differentially expressed proteins. Analyses revealed that transthyretin, a carrier protein for thyroid hormones and retinoids, and the brain form of phosphoglycerate mutase, a glycolytic enzyme, were upregulated in the cobalt-treated mouse cerebrum and further increased by PR administration in association with upregulation of neurogranin/RC3, a target of the transcriptional activation by thyroid hormones and retinoids. These findings suggest that PR-induced protection of mouse cerebral neurons involves neurotrophic events caused by thyroid hormones and/or retinoids and enhanced glycolysis.

Tissue distribution and hepatic subcellular distribution of perfluorooctanoic acid at low dose are different from those at high dose in rats.

Fate of perfluorooctanoic acid (PFOA) after an intravenous injection to male rats at the dose of 0.041 mg/kg body weight was compared with that at the dose of 16.56 mg/kg body weight. In the liver, 52% and 27% of PFOA dosed was recovered 2 h after an intravenous injection at the low and the high doses, respectively. By contrast, larger proportion of PFOA dosed was distributed to serum, other tissues and carcass at the high dose compared with the low dose. Subcellular distribution of PFOA was determined in the liver. At the dose of 0.041 mg/kg, 45%, 34%, 18% and 3% were distributed to 8000 g pellet, 18000g pellet, 105000g pellet and 105000g supernatant fraction, respectively; 28%, 17%, 13% and 43% of PFOA were distributed to these fractions, respectively, at the dose of 16.56 mg/kg. The higher the concentration of hepatic PFOA was, the more the PFOA was distributed to 105000g supernatant fraction. Biliary excretion index increased as PFOA concentration raised in the liver. These results suggest that PFOA is preferentially taken-up by the liver, and distributed to membrane fractions, especially 18000g pellet, and hardly excreted into bile when exposed at very low dose.

Stearoyl-CoA desaturase activity is elevated by the suppression of its degradation by clofibric acid in the liver of rats.

A mechanism by which fibrates control stearoyl-CoA desaturase (SCD) in the liver was studied. Treatment of rats with 2-(4-chlorophenoxy)-2-methylpropionic acid (clofibric acid) or feeding of a fat-free diet markedly elevated hepatic activity of SCD. Both the treatment with clofibric acid and the feeding of the fat-free diet caused an increase in the steady-state level of SCD1 mRNA and enhanced transcriptional rate. The half-lives of SCD for control rats, rats treated with clofibric acid rats, and rats fed the fat-free diet were estimated to be 2.0, 3.9, and 1.9 h, respectively. Activity of palmitoyl-CoA chain elongase (PCE) was increased by both clofibric acid treatment and feeding of the fat-free diet as was observed with SCD. Steady-state level of rat fatty acid elongase 2 mRNA was increased by the treatment with clofibric acid or feeding of fat-free diet, although the transcriptional rate was not altered. Different from SCD, PCE was highly stable and its half-life was not changed by either clofibric acid or fat-free diet. These results strongly suggest that the decreased degradation of SCD is responsible for the increase in its activity in addition to increased transcription of SCD1 in the rats treated with clofibric acid.

Inhibitory effects of peony root extract on the large conductance calcium-activated potassium current essential in production of bursting activity.

To elucidate the mechanism of inhibitory action of peony root extract on pentylenetetrazol-induced bursting activity, effects of peony root extract on the iberiotoxin-sensitive large conductance calcium-activated potassium (BKCa) current that plays an essential role in the production of bursting activity were investigated. Peony root extract showed a clear inhibitory effect on the iberiotoxin-sensitive calcium-activated potassium current. Peony root extract also showed clear inhibitory effects on spontaneous bursting activity and BKCa current in the cerebral cortical neurons of the EL mouse, a hereditary epilepsy animal model. These results together with our previous studies, including the protective effect against neuron damage, indicate that peony root extract is a promising herbal drug for inhibition of convulsions.

Cytotoxic activity toward KB cells of 2-substituted naphtho[2,3-b]furan-4,9-diones and their related compounds.

We investigated the cytotoxic activity of 2-substituted naphtho[2,3-b]furan-4,9-diones. We have previously synthesized 33 types of 2-substituted and related compounds, and the cytotoxic activity of these compounds was then examined by a KB cell culture assay. 2-(3-Furanoyl)benzoic acids and 1,4-naphthoquinones had no activity. 2-Acetyl-4,9-dimethoxynaphtho[2,3-b]furan 4 showed low activity. However, parent naphtho[2,3-b]furan-4,9-dione 2 and most 2-substituted derivatives exhibited cytotoxic activity. The parent structure was therefore for cytotoxicity. 2-Formylnaphtho[2,3-b]furan-4,9-dione 11 had particularly potent activity (ED50=0.09 microg/ml).

Induction of tumor-specific cytotoxicity and apoptosis by doxorubicin.

Doxorubicin (adriamycin), an anthracycline antibiotic, showed higher cytotoxic activity against human tumor cell lines (oral squamous cell carcinoma HSC-2, HSC-3, submandibular gland carcinoma HSG, promyelocytic leukemia HL-60) than against normal human cells (gingival fibroblast HGF, pulp cell HPC, periodontal ligament fibroblast HPLF). Doxorubicin activated caspases 3, 8 and 9 in both HSC-2 and HL-60 cells, but induced internucleosomal DNA fragmentation only in HL-60 cells. Western blot analysis showed that doxorubicin did not significantly change the intracellular concentration of Bcl-2, Bax and Bad in HL-60 cells. Real-time PCR analysis showed that HPC cells expressed the highest amount of mdr1 mRNA, followed by HSC-2 > HGF > HSC-3 > HPLF > HSG > HL-60. ESR spectroscopy showed that doxorubicin produced no discernible radical under alkaline conditions (pH 7.4 to 10.5) except at pH 12.5, and it did not scavenge O2-, NO and DPPH radicals. The present study demonstrates that doxorubicin induces the tumor-specific cytotoxicity and some, but not all, apoptosis markers possibly by a radical-independent mechanism, and that mdr1 expression in the tumor cells is not related to the tumor specificity of doxorubicin.

Molecular mechanism of preventive effect of peony root extract on neuron damage.

The molecular mechanism of the protective effects of peony root extract and its component substances on neuron damage induced by the cobalt focus epilepsy model and the EL mouse was investigated. Long-term administration of peony root extract for 30 days prior to metallic cobalt powder application to the cerebral cortex of mice resulted in increased expression of A20, an inhibitor gene of cell death. In the EL mouse, a hereditary epilepsy animal model with vulnerable neurons, increased expression of A20 was observed even without administration of peony root extract. Long-term administration of peony root extract to the EL mouse resulted in a marked increase of expression of A20. These results suggested that an increase in A20 expression is the main molecular mechanism of protective action of peony root extract on neuron damage.

Mechanisms of gastric mucus secretion from cultured rat gastric epithelial cells induced by carbachol, cholecystokinin octapeptide, secretin, and prostaglandin E2.

The effects of carbachol, cholecystokinin octapeptide (CCK-8), secretin, prostaglandin E2 (PGE2), and second mediator-like substances (A23187, phorbol 12-myristate 13-acetate, and dibutyryl cAMP) on mucus secretion from cultured gastric epithelial cells were investigated. Gastric mucus was measured by an enzyme-linked lectin assay with soybean agglutinin and wheat germ agglutinin. Intracellular cAMP and Ca2+ were measured with a cAMP assay kit and an image analysis system using fura-2-loaded cells, respectively. Secreted mucus induced by any combination of receptor agonists was almost equal to the summation of each stimulated mucus secretion. On the other hand, combined stimulation with second mediator-like substances secreted mucus synergistically. These results suggest the existence of interactions among receptors for mucus secretion. Based on these results, the secretagogue induced intracellular cAMP and free calcium ([Ca2+]i) levels were measured in cultured gastric epithelial cells incubated with secretagogues. Secretin and PGE2 induced cAMP accumulation, and carbachol and CCK-8 induced a [Ca2+]i increase. To confirm these results, the effects of protein kinase A and C inhibitors and intracellular calcium chelator on mucus secretion were investigated. An intracellular calcium chelator inhibited the mucus secretion induced not only by carbachol and CCK-8 but also by secretin and PGE2. These results suggest that the [Ca2+]i plays an important role in mucus secretion through cAMP accumulation.