Bikaverin and fusaric acid from Fusarium oxysporum show antioomycete activity against Phytophthora infestans.

AIMS: To isolate and identify antioomycete substances from Fusarium oxysporum EF119 against Phytophthora infestans and to investigate their antimicrobial activities against various plant pathogenic bacteria, oomycetes and true fungi. METHODS AND RESULTS: Two antioomycete substances were isolated from liquid cultures of F. oxysporum EF119, which shows a potent disease control efficacy against tomato late blight caused by P. infestans. They were identified as bikaverin and fusaric acid by mass and nuclear magnetic resonance spectral analyses. They inhibited the mycelial growth of plant pathogenic oomycetes and fungi. Fusaric acid also effectively suppressed the cell growth of various plant pathogenic bacteria, but bikaverin was virtually inactive. Treatment with bikaverin at 300 microg ml(-1) suppressed the development of tomato late blight by 71%. Fusaric acid provided effective control against tomato late blight and wheat leaf rust over 67% at concentrations more than 100 microg ml(-1). CONCLUSIONS: Both bikaverin and fusaric acid showed in vitro and in vivo antioomycete activity against P. infestans. SIGNIFICANCE AND IMPACT OF THE STUDY: Fusarium oxysporum EF119 producing both bikaverin and fusaric acid may be used as a biocontrol agent against tomato late blight caused by P. infestans.

Simultaneous screen for microsomal stability and metabolite profile by direct injection turbulent-laminar flow LC-LC and automated tandem mass spectrometry.

A LC-LC/MS/MS method has been developed that significantly increases the throughput in metabolism screening of drug candidates during lead optimization in discovery. This was accomplished by the reduction of sample preparation time through an on-line extraction of a drug and its metabolites from microsomal proteins using turbulent flow chromatography. Following its injection onto a column at turbulent flow, the drug and its metabolites are backwashed onto a reverse-phase column via on-line column switching and resolved chromatographically at a laminar flow of 2 mL/min. This tandem turbulent-laminar flow chromatographic system in a total cycle time of 8 min can achieve adequate separation of isomeric metabolites of venlafaxine, haloperidol, or adatanserin. Further improvement in throughput can be achieved by multiplexing both microsomal stability assessment and metabolite profiling into a single analysis. This is made possible by the ability of the ion-trap mass spectrometer to perform simultaneously multiple-reaction monitoring for microsomal stability and data-dependent multiple-stage mass spectrometric analysis for metabolite profiling within a single LC analysis. Such a LC-LC/MS/MS approach can dramatically shorten the time for providing metabolism feedback to the drug discovery process.

Protective effects of acetylbergenin against carbon tetrachloride-induced hepatotoxicity in rats.

The present study was undertaken to investigate whether or not the hepatoprotective activity of acetylbergenin was superior to bergenin in carbon tetrachloride (CCl4)-intoxicated rat. Acetylbergenin was synthesized by acetylating bergenin, which was isolated from Mallotus japonicus. The hepatoprotective effects of acetylbergenin were examined against CCl4-induced liver damage in rats by means of serum and liver biochemical indices. Acetylbergenin was administered orally once daily for 7 successive days, then a 0.5 ml/kg mixture of CCl4 in olive oil (1:1) was intraperitoneally injected at 12 h and 36 h after the final administration of acetylbergenin. Pretreatment with acetylbergenin reduced the elevated serum enzymatic activities of alanine/aspartate aminotransferase, sorbitol dehydrogenase and gamma-glutamyltransferase in a dose dependent fashion. Acetylbergenin also prevented the elevation of hepatic malondialdehyde formation and depletion of glutathione content dose dependently in CCl4-intoxicated rats. In addition, the decreased activities of glutathione S-transferase and glutathione reductase were restored to almost normal levels. The results of this study strongly suggest that acetylbergenin has potent hepatoprotective activity against CCl4-induced hepatic damage in rats by glutathione-mediated detoxification as well as having free radical scavenging activity. In addition, acetylbergenin doses of 50 mg/kg showed almost the same levels of hepatoprotective activity as 100 mg/kg of bergenin, indicating that lipophilic acetylbergenin is more active against the antihepatotoxic effects of CCl4 than those of the much less lipophilic bergenin.

A method for the simultaneous evaluation of the activities of seven major human drug-metabolizing cytochrome P450s using an in vitro cocktail of probe substrates and fast gradient liquid chromatography tandem mass spectrometry.

A method for the simultaneous evaluation of the activities of seven major human drug-metabolizing cytochrome P450s (CYP3A4, CYP2D6, CYP2C9, CYP1A2, CYP2C19, CYP2A6, and CYP2C8) was developed. This method uses an in vitro cocktail of specific substrates (midazolam, bufuralol, diclofenac, ethoxyresorufin, S-mephenytoin, coumarin, and paclitaxel) and fast gradient liquid chromatography tandem mass spectrometry. The assay incubation time is 20 min, which is in the linear range for all of the substrates, and the analysis time is 4 min/sample. Substrate specificity was confirmed by incubating Escherichia coli-expressed enzymes with the cocktail. Potent specific inhibitors of the seven enzymes (ketoconazole, quinidine, sulfaphenazole, tranylcypromine, quercetin, furafylline, and 8-methoxypsoralen) were evaluated in cocktail and individual substrate incubations. Five of these inhibitors were further studied to determine more precise IC(50) values for inhibition of the seven enzymes. The IC(50) values obtained in both cocktail and individual incubations were in good agreement with published values. This cocktail method offers an efficient, robust way to determine the cytochrome P450 inhibition profile of large numbers of compounds. The enhanced throughput of this method greatly facilitates its use to assess CYP inhibition as a drug candidate selection criterion.

Hepatoprotective effects of bergenin, a major constituent of Mallotus japonicus, on carbon tetrachloride-intoxicated rats.

The hepatoprotective effects of bergenin, a major constituent of Mallotus japonicus, were evaluated against carbon tetrachloride (CCl(4))-induced liver damage in rats. Bergenin at a dose of 50, 100 or 200 mg/kg was administered orally once daily for successive 7 days and then a mixture of 0.5 ml/kg (ip) of CCl(4) in olive oil (1:1) was injected two times each at 12 and 36 h after the final administration of bergenin. The substantially elevated serum enzymatic activities of alanine/aspartate aminotransferase, sorbitol dehydrogenase and gamma-glutamyltransferase due to CCl(4) treatment were dose dependently restored towards normalization. Meanwhile, the decreased activities of glutathione S-transferase and glutathione reductase were restored towards normalization. In addition, bergenin also significantly prevented the elevation of hepatic malondialdehyde formation and depletion of reduced glutathione content in the liver of CCl(4)-intoxicated rats in a dose dependent fashion. The results of this study clearly indicate that bergenin has a potent hepatoprotective action against CCl(4)-induced hepatic damage in rats.

Protective effects of bergenin, the major constituent of Mallotus japonicus, on D-galactosamine-intoxicated rat hepatocytes.

This study was designed to investigate the effects of bergenin against D-galactosamine-induced injury in primary cultured rat hepatocytes. Bergenin (100 microM) decreased the release of glutamic pyruvic transaminase and sorbitol dehydrogenase by 62 and 50%, respectively, into hepatocyte medium incubated for 14 h with 1.5 mM galactosamine. Decreased RNA synthesis by 1.5 mM galactosamine was recovered 2.5 times compared with that of control hepatocytes at 100 microM bergenin. Therefore, the present results suggest that bergenin show hepatoprotective effects against galactosamine-intoxicated rat hepatocytes by inhibiting the release of glutamic pyruvic transaminase and sorbitol dehydrogenase as well as by increasing RNA synthesis.

Anti-inflammatory effects of fangchinoline and tetrandrine.

Fangchinoline and tetrandrine are the major alkaloids from Stephania tetrandrae S. Moore which has been used traditionally for the treatment of inflammatory diseases in oriental countries including Korea. Both fangchinoline and tetrandrine showed anti-inflammatory effects on mouse ear edema induced by croton oil. In addition, the effects of fangchinoline and tetrandrine on cyclooxygenase, murine interleukin-5 (mIL-5) and human interleukin-6 (hIL-6) were examined in vitro to investigate the anti-inflammatory action mechanisms. One hundred micromolar of fangchinoline showed 35% of inhibition on cyclooxygenase, but the same concentration of tetrandrine did not show any inhibition. On the other hand, 12.5 microM of tetrandrine exhibited 95% of inhibition on mIL-5 activity, while fangchinoline did not show any effects. However, 4 microM of fangchinoline and 6 microM of tetrandrine showed 63 and 86% of inhibitions on hIL-6 activity, respectively. These results suggest that biochemical mechanisms of fangchinoline and tetrandrine on anti-inflammation are significantly different even though they are similar in chemical structure.

Antihepatotoxic activity of bergenin, the major constituent of Mallotus japonicus, on carbon tetrachloride-intoxicated hepatocytes.

To determine the antihepatotoxic activity of bergenin from Mallotus japonicus, carbon tetrachloride (CCl4)-induced cytotoxicity in primary cultured rat hepatocytes has been adopted as an assay system. Bergenin significantly reduced the activities of glutamic pyruvic transaminase and sorbitol dehydrogenase released from the CCl4-intoxicated hepatocytes. The antihepatotoxicity of bergenin was also evidenced by elevating the activities of glutathione S-transferase and glutathione reductase, and content of glutathione in the CCl4-intoxicated hepatocytes. From these results, it is assumed that bergenin exerted antihepatotoxicity against CCl4-induced cytotoxicity through glutathione-mediated detoxification as well as free radical suppressing activity.

Inhibitory effects of velvet antler water extract on morphine-induced conditioned place preference and DA receptor supersensitivity in mice.

The repeated administration of morphine in mice produced conditioned place preference (CPP) and postsynaptic dopamine (DA) receptor supersensitivity. CPP effects were evaluated assessing the increased time spent by the mice to morphine and the inhibition of CPP by the decreased time spent by the mice in the nonpreferred compartment. Postsynaptic DA receptor supersensitivity in mice displaying a morphine-induced CPP was evidenced by the enhanced response in ambulatory activity to the DA agonist. The oral administration of velvet antler water extract (VAWE) from Cerrus elaphus, prior to the morphine treatment in mice inhibited the development of morphine-induced CPP and postsynaptic DA receptor supersensitivity. These results suggest that the inhibitory effects of VAWE on morphine-induced CPP may be closely related to the recovery of dysfunction in the dopaminergic system produced by morphine because both phenomena were inhibited by VAWE. But a single administration of VAWE did not inhibit apomorphine-induced climbing behavior, indicating that antidopaminergic activity was not produced by a single administration of VAWE at the postsynaptic DA receptors. From the above results, it is presumed that VAWE may be useful for prevention and therapy of the adverse actions of morphine.

Antinarcotic effects of the velvet antler water extract on morphine in mice.

The present study was undertaken to investigate the antinarcotic effects of velvet antler water extract (VAWE) from Cervus elaphus on morphine. Morphine-induced analgesic action was measured by tail-flick method. Morphine-induced hyperactivity and reverse tolerance were evidenced by measuring the enhanced ambulatory activity using a tilting-type ambulometer. Dopamine (DA) receptor supersensitivity in mice displaying morphine-induced reverse tolerance was evidenced by the enhanced response in ambulatory activity to the DA agonist, apomorphine. The repeated administration of VAWE significantly inhibits the development of morphine-induced analgesic tolerance, physical dependence, reverse tolerance and postsynaptic DA receptor supersensitivity. But a single administration of VAWE did neither antagonize morphine-induced analgesia nor inhibit morphine-induced hyperactivity. From the above results, it is presumed that VAWE may be useful for prevention and therapy of the adverse actions of morphine caused by the repeated administration of morphine.

Iodized oil retention due to postbiopsy arterioportal shunt: a false positive lesion in the investigation of hepatocellular carcinoma.

BACKGROUND: Iodized-oil computed tomography (CT) is useful for the diagnosis of hepatocellular carcinoma, but there may be false-positive results in patients who have undergone some percutaneous transhepatic interventional procedures. The purpose of the present study was to verify the correlation between subsegmental hepatic parenchymal retention of iodized oil on CT and the arterioportal shunt caused by liver biopsy. METHODS: Iodized-oil CT scans were reviewed in 24 patients with hepatic tumors who had liver biopsy and subsequent iodized oil chemoembolization. Iodized oil chemoembolization was performed shortly after biopsy (1-10 days; mean = 2.6 days). The results were correlated with hepatic arteriography, with a special emphasis on the presence of hepatic arterioportal shunt. RESULTS: Wedge-shaped subsegmental retention of iodized oil along or adjacent to the biopsy needle path was observed in iodized-oil CT in 17 of the 24 patients. In three patients, there was subsegmental enhancement on prebiopsy helical dynamic liver CT at the same area of iodized oil retention, and therefore iodized oil retention was considered to be due to hepatocellular carcinoma. In the remaining 13 (54%) patients, the peripheral iodized oil retention was considered to be due to biopsy-induced arterioportal shunt. In all these patients, arterioportal shunt was confirmed by hepatic arteriography. CONCLUSION: Wedge-shaped hepatic parenchymal retention of iodized oil is commonly observed in iodized-oil CT due to biopsy-induced arterioportal shunt, and this appearance should not be confused with a hepatocellular carcinoma.

Appendicitis: usefulness of color Doppler US.

PURPOSE: To assess whether color Doppler ultrasonography (US) can help diagnose early acute appendicitis in appendices of equivocal size at gray-scale US. MATERIALS AND METHODS: In a retrospective study, color Doppler US findings were evaluated in 20 individuals with proved normal appendix at barium enema examination and in 50 patients in whom pathologic findings confirmed acute appendicitis. In a prospective study, the diagnostic value of hyperemia in the wall of the appendix in differentiating a normal from an inflamed appendix was evaluated in 26 patients with appendices of equivocal size (5-7 mm in maximal outer diameter) at grayscale US. RESULTS: In the retrospective study, 44 of the 50 patients had hyperemia at color Doppler US. The remaining six patients had no hyperemia, and pathologic findings confirmed gangrenous appendices. The 20 patients with proved normal appendix at barium enema examination had no detectable blood flow in the wall. In the prospective study, 10 of 26 patients had hyperemia at color Doppler US, and pathologic findings confirmed early acute appendicitis. Results of barium enema examination (n = 12) or clinical follow-up (n = 4) confirmed no appendicitis in the remaining 16 patients. CONCLUSION: Hyperemia in the wall of the appendix at color Doppler US is a sensitive indicator for inflammation. A simple additional color Doppler US examination may be helpful in the diagnosis of early acute appendicitis when an appendix is well depicted and is equivocal in size at gray-scale US.

Assessment of reducibility of ileocolic intussusception in children: usefulness of color Doppler sonography.

PURPOSE: To assess the diagnostic usefulness of ultrasonography (US) in intussusception in children and determine whether color Doppler sonography (CDS) can indicate bowel viability and help in prediction of reducibility. MATERIALS AND METHODS: US was used to examine 176 children in whom the presence of intussusception was clinically suspected. CDS was performed in 64 patients with 65 cases of intussusception proved with US. RESULTS: The positive US findings were confirmed with air enema; the negative findings, with air enema in 46 patients and clinical follow-up in 66 patients (100% specificity and sensitivity). CDS indicated blood flow in 62 of 65 cases of intussusception; pneumatic reduction was achieved in 58 (94%) of these 62 cases. Four patients (6%) underwent manual reduction of viable bowel; three patients (5%) who had no color Doppler signal underwent segmental resection. The difference in reduction rates based on blood flow was statistically significant (P = .0008). CONCLUSION: US is sensitive and specific in the diagnosis of intussusception, and CDS is a promising predictor of bowel viability.