Inhibition of aldehyde reductase by aldose reductase inhibitors

A broad group of structurally diverse aldose reductase inhibitors including flavonoids, carboxylic acids and hydantoins, have been examined for their ability to inhibit rat kidney aldehyde reductase (EC 1.1.1.19, EC 1.1.1.20) versus rat lens aldose reductase (EC 1.1.1.21). All aldose reductase inhibitors examined inhibited aldehyde reductase to some extent both in the reductive reaction as determined with glyceraldehyde as substrate and NADPH as coenzyme, and in the oxidative reaction where L-gulonic acid was oxidized to D-glucuronic acid in the presence of NADP+. Of the inhibitors examined, 2,7-difluorospirofluorene-9,5'-imidazolidine-2',4'-dion e (Al1576) was the most potent inhibitor requiring only concentrations in the 10(-8) M range to inhibit 50% of the in vitro activity of rat kidney aldehyde reductase (IC50 value), whereas 3-dioxo-1-H-benz[de]isoquinoline-2(3H)-acetic acid (alrestatin) was the least potent inhibitor requiring concentrations in the 10(-5) M range. Both the reductive and oxidative steps appeared equally inhibited by these aldose reductases inhibitors. Moreover, all compounds appeared to inhibit either crude or highly purified rat kidney aldehyde reductase to essentially the same extent. Marked differences in the selectivity of these inhibitors, expressed as the ratio of IC50 values for rat kidney aldehyde reductase versus rat lens aldose reductase with glyceraldehyde as substrate, were observed with selectivity for aldose reductase ranging from ca. 2-fold for Al1576 to 119-fold for 3-(4-bromo-2-fluorobenzyl-4-oxo-3-phthalazine-1-ylacetic acid (Ponalrestat). Kinetic and competition studies suggest that these inhibitors interact with aldehyde reductase at a common site that is not identical to either the substrate or nucleotide binding site. These results suggest that the inhibitor binding sites of rat kidney aldehyde reductase and aldose reductase contain several common characteristics.     

Inhibition of microsomal cytochrome c reductase activity by a series of alpha, beta-unsaturated aldehydes

alpha, beta-Unsaturated aldehydes are reactive and cytotoxic compounds which occur in the environment and are also formed in vivo. Many of these aldehydes have been reported to inhibit hepatic cytochrome P-450. Our laboratory has shown that trans,trans-muconaldehyde (a possible metabolite of benzene) as well as acrolein and crotonaldehyde, when added to hepatic microsomes, decreased cytochrome P-450 (measured spectrophotometrically). Additional studies showed that several alpha, beta-unsaturated aldehydes also inhibited hepatic microsomal NADPH-cytochrome c reductase. Acrolein, crotonaldehyde and trans,trans-muconaldehyde all decreased NADPH-cytochrome c reductase activity in vitro. Concentrations of 0.5, 1.0 and 1.5 mM acrolein decreased activity to 60, 26 and 11% of control respectively. Similar concentrations of trans,trans-muconaldehyde inhibited NADPH-cytochrome c reductase. Crotonaldehyde was a less effective inhibitor of this enzyme. Propionaldehyde, a saturated aldehyde, had no effect on NADPH-cytochrome c reductase activity. Time course experiments with acrolein over a period of 5-45 min suggest that the loss of NADPH-cytochrome c reductase activity is non-linear. The addition of reduced glutathione protected against the inhibition of reductase activity by acrolein. Formation of these aldehydes and their subsequent inhibition of these enzymes may have important consequences in xenobiotic metabolism.     

Inhibition of rat folic acid reductase activity by trimethoprim during the later stages of gestation

Summary The increased level of folic acid reductase activity in rats during the perinatal period is inhibited following the oral administration of 5 or 50 mg/kg Trimethoprim. When the enzyme activity was tested in vitro, the highest sensitivity to the antimetabolite was displayed by the liver reductase isolated from rats the 20th day of pregnancy, the lowest was observed in the foetal liver extract.It is proposed that the stimulated reductase activity during the pregnancy is caused by a newly synthesized, Trimethoprim-sensitive enzyme form. The results of the in vitro experiments could contribute to the suggested hypothesis.     

Inhibition of cellular thioredoxin reductase by diaziquone and doxorubicin. Relationship to the inhibition of cell proliferation and decreased ribonucleotide reductase activity.

The flavoenzyme thioredoxin reductase (TR) is an important enzyme for many aspects of cellular function. The antitumor quinones diaziquone and doxorubicin have been shown to produce a time- and concentration-dependent inhibition of TR when incubated for up to 24 hr with intact A204 human rhabdomyosarcoma cells. There was a positive correlation between the inhibition of TR and the inhibition of cell colony formation measured 7 days later for diaziquone (r = 0.84, P less than 0.01), and for doxorubicin (r = 0.87, P less than 0.01). 2,6-Dichloroindophenol, which in previous studies was shown to be a good inhibitor of TR in vitro, was a poor inhibitor of TR in intact A204 cells and there was no significant correlation with inhibition of colony formation. The activity of ribonucleotide reductase, which catalyzes the first unique step of DNA synthesis and which obtains its reducing equivalents from TR through thioredoxin, was decreased in diaziquone- and doxorubicin treated A204 cells. We suggest that the inhibition of TR by some antitumor quinones leading to a decreased activity of TR and, consequently, a decreased activity of thioredoxin-dependent enzymes including ribonucleotide reductase may contribute to the growth inhibitory activity of these quinones.     

Inhibition of cholesterol absorption by HMG-CoA reductase inhibitor

Summary In subjects with familial hypercholesterolemia cholesterol absorption efficiency was insignificantly reduced during a short-term more consistently during longterm pravastatin treatment. A cholesterol feeding had no effect on LDL cholesterol level but reduced absorption efficiency during a long-term lovastatin treatment.     

Anti-Propionibacterium acnes assay-guided purification of brazilin and preparation of brazilin rich extract from Caesalpinia sappan heartwood

Context: Caesalpinia sappan L. (Leguminosae or Fabaceae) heartwood has been used as a coloring agent, with antibacterial activity in food, beverages, cosmetics, and garments.

Objectives: To purify brazilin from C. sappan heartwood and use it as a standard marker for the preparation and standardization of an active constituent-rich extract.

Material and methods: Crude ethanol extracts of C. sappan heartwood (CSE) were fractionated to isolate brazilin by an anti-P. acnes assay-guided isolation. Quantitative analysis was performed by HPLC. Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) were determined by the broth microdilution method.

Results and discussion: Brazilin isolated from CSE possessed antibacterial activity against P. acnes with MIC and MBC values of 15.6 and 31.2?µg/mL, respectively. Brazilin was, therefore, used as a standard marker for standardization and preparation of a brazilin rich extract (BRE). BRE was prepared from CSE using a simple one-step purification using a macroporous resin column eluted with 35% v/v ethanol. This method increased the brazilin content in the BRE up to 39.9% w/w. The antibacterial activity of the standardized BRE against acne involved bacteria was higher than for the CSE but lower than brazilin. However, for industrial applications, a large-scale one-step preparation of BRE has more advantages than the use of pure brazilin in terms of convenience and a low-cost production process. Therefore, BRE is considered as a potential coloring agent with antibacterial activity which is used for pharmaceutical, cosmetic, and nutraceutical applications.     

Inhibition of rabbit heart carbonyl reductase by fatty acids.

The inhibition of rabbit heart carbonyl reductase (RHCR) by fatty acids was examined using 4-benzoylpyridine (4BP) as a substrate. The inhibitory potency of saturated fatty acids increased with elongation in the carbon chain from caprylic acid to myristic acid, but decreased with further elongation. Myristic acid with 14 carbon atoms most strongly inhibited RHCR. All of the unsaturated fatty acids tested strongly inhibited RHCR; the cis-isomers were more potent inhibitors than the corresponding trans-isomers. The methyl esters and alcohols, which lack a carboxyl group, derived from fatty acids did not exert a significant inhibitory effect on RHCR. These results indicate that the existence of a proper length of carbon chain, double bond(s), and a carboxyl group in a fatty acid molecule is important for RHCR inhibition. We also propose the possibility that myristic acid at low concentrations inhibits the reduction of 4BP by interacting with a binding site other than the coenzyme- and substrate-binding sites of RHCR.     

Inhibition of ribonucleotide reductase by antitumor agents related to levodopa and dopamine

Using partially purified enzyme from L1210 cells, dihydroxybenzene derivatives related structurally to dopamine were shown to reversibly inactivate ribonucleotide reductase. A structure-activity analysis revealed that derivatives with side-chains, which contain a negatively-charged group, had significantly reduced inhibitory activity. The ability of these compounds to inhibit ribonucleotide reductase was dependent on the hydroxyl groups being in the ortho position and did not correlate with free radical inhibitory activity. A kinetic analysis by the method of Lineweaver-Burk indicated that the inhibition of ribonucleotide reductase by the derivative 3,4-dihydroxybenzylamine was competitive with the reducing substrate dithioerythritol. This analog, in combination with hydroxyurea, gave synergistic inhibition or ribonucleotide reductase, suggesting different sites of action. Using Tween 80-treated L1210 cells, it was found that these drugs had an immediate inhibitory effect on ribonucleotide reductase activity in intact, reversibly permeabilized cells. Furthermore, although these drugs had no immediate effect on DNA polymerase, in permeabilized L1210 cells (when the cells were preincubated with the dihydroxybenzene derivatives for 1 hr prior to permeabilization), there was significant inhibition of DNA polymerase activity. The two key enzymes for DNA synthesis appear to be sequentially inhibited by these analogs, with the reduced form (quinol) inhibiting ribonucleotide reductase and the oxidized form (quinone) inhibiting DNA polymerase.     

Inhibition of human lens aldose reductase by flavonoids,sulindac and indomethacin

The inhibition of human lens aldose reductase by flavonoids has been studied. Quercetin, the major pentahydroxyflavone, was observed to inhibit human lens aldose reductase by 50% at a concentration of 5 × 10^?6 M. The inhibitory activity of its 3-O-glucoside was similar to that of the parent aglycon. Glycosidation with l-sugar (quercitrin and guaijaverin), however, inproved the inhibitory activity (the IC₅₀ values being 1 × 10^?6 M and 2.5 × 10^?6 M respectively). The improvement in inhibitory activity with glycosidation with l-sugar was also apparent from the high inhibitory activity of myricitrin as compared to myricetin, although the improvement in this case of hexahydroxy flavone glycosidation was significantly less than in the case of penthahydroxy flavone glycosidation. The structure-activity relationship observed for human lens enzyme was similar to that reported previously for rat lens enzyme. Inhibitory activity on the whole however, was lower with human lens enzyme. Some known inhibitors of cyclo-oxygenase such as indomethacin, aspirin and sulindac also inhibited human lens aldose reductase. Thus, an inhibitor of one of the enzymes may actually inhibit both and. when administered, may exert mixed physiological effects.     

Inhibition of ribonucleotide reductase and L1210 cell growth by N-hydroxy-N'-aminoguanidine derivatives

A series of N-hydroxy-N'-aminoguanidine derivatives was studied for their effects on L1210 cell growth and ribonucleotide reductase activity. With the twelve compounds studied, there was a good correlation between the inhibition of L1210 cell growth and the inhibition of ribonucleotide reductase activity. The most potent compound required concentrations of only 1.4 and 2 microM for 50% inhibition of L1210 cell growth and ribonucleotide reductase activity respectively. These guanidine analogs specifically inhibited the conversion of [14C]cytidine and deoxycytidine nucleotides in the nucleotide pool and the incorporation of [14C]cytidine into DNA without altering the incorporation of [14C]cytidine into RNA. Ribonucleotide reductase activity in drug-treated cells was reduced markedly. Iron-chelating agents did not either increase or decrease the inhibition caused by the N-hydroxy-N'-aminoguanidine derivatives. No evidence was obtained that these derivatives selectively inactivated one of the subunits of ribonucleotide reductase. These compounds appear to inhibit ribonucleotide reductase by a mechanism different from hydroxyurea or the thiosemicarbazone derivatives.     

Inhibition of cholinesterase activity by isocyanates

Exposure of workers to isocyanates may result in irritation and/or sensitization of the respiratory tract. An immunologic mechanism for sensitization has been presented previously. This investigation explored whether, as a possible mechanism for the irritation reaction, the toxic respiratory effect of isocyanates might be due to their ability to inhibit cholinesterases. Hexamethylene diisocyanate (HDI), hexyl isocyanate (HI), and 2,6-toluene diisocyanate (2,6-TDI) were found to completely inhibit purified human serum cholinesterase when added at molar ratios of 4:1 to 8:1 (isocyanate:enzyme). By contrast, molar ratios of 50:1 or greater were required for 50% enzyme inhibition by 2,4-toluene diisocyanate (2,4-TDI), phenyl isocyanate, or o-tolyl isocyanate. Enzyme inhibition was also achieved by exposure of purified cholinesterase to atmospheres containing 1 ppm isocyanates. Under these conditions, HDI and HI were again the most potent enzyme inhibitors with much less reactivity shown by 2,4-TDI and 2,6-TDI. Under more physiologic conditions, when whole human plasma was the source of cholinesterase, HDI and HI were still potent enzyme inhibitors. However, with the latter two isocyanates, the molar concentrations needed to effect 50% enzyme inhibition suggested affinity labeling by these reagents. The potent cholinesterase inhibition shown by HDI and HI may offer explanation for observed respiratory symptomatology noted upon exposure to these isocyanates.     

Inhibition of aromatase activity by flavonoids

In searching for potent cancer chemopreventive agents from synthetic or natural products, 28 randomly selected flavonoids were screened for inhibitory effects against partially purified aromatase prepared from human placenta. Over 50% of the flavonoids significantly inhibited aromatase activity, with greatest activity being demonstrated with apigenin (IC50: 0.9 microg/mL), chrysin (IC50: 1.1 microg/mL), and hesperetin (IC50: 1.0 microg/mL).     

HPLC测定苏木中的巴西苏木素和原苏木素B

目的 建立HPLC测定苏木中巴西苏木素和原苏木素B含量的方法.方法 色谱柱为Agilent Zorbax XDB C18(150 mmx4.6 ,5μm),流动相为甲醇-0.1%醋酸(20:80),流速为1.0 mL·min-1,检测波长为280 nm.结果 巴西苏木素和原苏木素B的线性范围均为0.025～0.50 mg·mL-1,r均为0.9994;平均同收率分别为95.44%(RSD=2.57%)和94.69%(RSD=1.38%).结论 新建方法简单、快速、准确,适用于苏木中巴西苏木素和原苏木素B的含量测定.