An isoquinoline alkaloid, berberine, can inhibit fungal alpha amylase: enzyme kinetic and molecular modeling studies

Aspergillus flavus is a commonly found fungal pathogen, which produces aflatoxins, highly toxic and hepatocarcinogenic natural compounds. Inhibition of fungal alpha amylase activity has been found to limit the ability of the fungus to produce aflatoxins. Berberine, an isoquinoline alkaloid commonly found in many medicinal plants, was identified to inhibit the growth of A. flavus. The amount of berberine required to inhibit the fungal mycelial growth was determined. The compound was also found to inhibit the alpha amylase from the A. flavus. The binding affinity of the compound toward alpha amylase and the enzyme inhibitory activity have been determined by enzyme kinetic studies and Isothermal Titration Calorimetric analysis. Molecular modeling and docking studies were carried out to understand the enzyme–ligand interactions.     

In vitro antioxidant activity of piperine

Oxygen radical injury and lipid peroxidation have been suggested as major causes of atherosclerosis, cancer, liver disease and the aging process. Piperine, having an antiinflammatory effect, has been demonstrated in in vitro experiments to protect against oxidative damage by inhibiting or quenching free radicals and reactive oxygen species and hydroxyl radicals. The effect on lipid peroxidation was also examined and IC50 values were calculated. Piperine was found to act as a hydroxyl radical scavenger at low concentrations, but at higher concentrations, it activated the fenton reaction resulting in increased generation of hydroxyl radicals. Whereas it acts as a powerful superoxide scavenger and IC50 is 1.82 mM, a 52% inhibition of lipid peroxidation was observed at a dose of 1400 microM with an IC50 of 1.23 mM. The results depict that piperine possesses direct antioxidant activity against various free radicals. This study also opens newer views on the potential efficacy of piperine in protecting tissues from peroxidative damage.     

In vitro anti-influenza virus activity of isoquinoline alkaloids from thalictrum species

The in vitro anti-influenza virus effects of some isoquinoline alkaloids, isolated from Thalictrum species (Ranunculaceae), growing in Mongolia and Sweden have been studied. (-)-thalimonine (Thl) and (-)-thalimonine N-oxide, isolated from the Mongolian plant T. simplex, inhibited markedly the influenza virus reproduction in vitro; thalictuberine N-oxide was less effective. The synthetic analogues of Thl as well as the rest of the tested alkaloids, isolated from T. foetidum and T. flavum, were not active. At a concentration range between 0.1 - 6.4 microM Thl inhibited viral reproduction in a selective and specific way.     

In vitro antiproliferative effects of the indole alkaloid vallesiachotamine on human melanoma cells

In course of a screening for small molecules presenting potential anticancer properties, a known monoterpene indole alkaloid named vallesiachotamine was isolated from the leaves of Palicourea rigida (Rubiaceae) collected in the Brazilian Cerrado. The structure was determined by spectroscopic methods, mainly 1D- and 2D-NMR and its biological activities were investigated on cultured human (SK-MEL-37) melanoma cells. In vitro cytotoxicity was evaluated by the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The inhibitory concentration (IC₅₀) was 14.7 ± 1.2 μM for 24 h of drug exposure. Flow cytometry analysis revealed that vallesiachotamine induced G0/G1 arrest and increased the proportion of sub-G1 hypodiploid cells (at 11 μM and 22 μM) and this effect was not dependent on time of incubation. At these concentrations, a typical ladder was observed by agarose gel electrophoresis of the extracted DNA. Treatment of cells with 50 μM vallesiachotamine for 24 h caused extensive cytotoxicity and necrosis. Our results demonstrated that the indole alkaloid vallesiachotamine exhibited important cytotoxicity toward human melanoma cells and that apoptosis and necrosis might be responsible for the observed events.     

In vitro studies on anthracycline haloderivatives

A new class of anthracycline derivatives carrying a halogen atom in the 4' position of the aminosugar moiety was tested in cytotoxicity studies on HeLa cells and on P388 cell lines sensitive and resistant (P388/DX) to doxorubicin, in comparison with the parent drugs doxorubicin (DX) and daunorubicin (DNR). 4'-Haloderivatives of DX generally appear to be more cytotoxic than DX on HeLa, P388 and P388/DX cells. Cellular kinetic studies of DX-haloderivatives on HeLa, P388 and P388/DX cell lines show that they accumulate inside the cell in higher amounts than DX, whereas DNR haloderivatives accumulate in HeLa and P388 cells at levels similar to DNR; only in P388/DX is their accumulation higher as compared with DNR. The results reported suggest that, besides drug accumulation, other factors are involved in the cytotoxic mechanism of action of this class of compounds. Therefore 4'-haloderivatives represent a class of compounds with promising activity, in particular regarding anthracycline-resistant cell lines.     

In vitro antioxidant properties of morphine.

Morphine is implicated in diverse functions, from development to immune modulation in the central and peripheral nervous systems. It has also been used extensively in the clinical management of pain due to its potent analgesic effect. This study was designed to evaluate the in vitro antioxidant capacity of morphine using different antioxidant tests, including total antioxidant activity, reducing power, free radical scavenging, superoxide anion radical scavenging, hydrogen peroxide scavenging and metal chelating activities. Morphine exhibited strong total antioxidant activity. The concentrations of 25, 50 and 75 microgml(-1) of morphine showed 79.1, 84.3 and 92.3% inhibition on peroxidation of linoleic acid emulsion, respectively. On the other hand, at 75 microgml(-1) concentration of standard antioxidant, such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and alpha-tocopherol, exhibited 88.7, 94.5 and 70.4% inhibition on peroxidation of linoleic acid emulsion, respectively. In addition, morphine had effective reducing power, free radical scavenging, superoxide anion radical scavenging, hydrogen peroxide scavenging and metal chelating activities at the same concentrations (25, 50 and 75 microgml(-1)). These various antioxidant activities were compared to standard antioxidants such as BHA, BHT and alpha-tocopherol.     

A new cytotoxic benzophenanthridine isoquinoline alkaloid from the fruits of Macleaya cordata

A new cytotoxic benzophenanthridine isoquinoline alkaloid, named cordatine (1), together with one known alkaloid 8-methoxydihydrochelerythrine (2), was isolated from the fruits of Macleaya cordata. The structure of the new compound was elucidated by spectroscopic methods including 1D and 2D NMR, HR-ESI-MS. Both compounds indicated significant cytotoxicity against MCF-7 and SF-268 cell lines.     

In vitro studies on the blood distribution of almitrine

Blood binding of almitrine, a highly lipophilic drug, was investigated in vitro. [3H]-Almitrine was incubated in a serum pool and isolated protein and lipoprotein fractions. The investigations were performed by using ultracentrifugation and another method which measures the uptake by proteins from glass beads coated with almitrine. Our results with ultracentrifugation show that the distribution of almitrine in serum takes place predominantly in the lipoprotein fraction (78%) and to a minor extent (22%) in the fraction of d greater than 1.20 (albumin-rich fraction). Experiments using glass beads coated with almitrine were then conducted to measure the binding of almitrine to isolated plasma proteins. The maximal uptake values (mol almitrine/mol lipoprotein) of almitrine by isolated lipoproteins decrease from VLDL (260) to LDL (20) to HDL (3) and seem to be related to the lipid content of the particles. The uptake by albumin and alpha 1-acid glycoprotein was low. The molar ratios of [almitrine]/[lipoprotein] are roughly proportional to almitrine concentrations within the therapeutic range. When almitrine was incubated in erythrocytes suspended in several dilutions of serum, almitrine partitioned less in erythrocytes as the serum protein concentration increased in the suspension.     

In vitro antioxidant activity of three Piper species

Reactive oxygen species are known to play a role in numerous pathological conditions, and antioxidants from plant sources have been of great help in modulating this effect. This study assesses the methanolic extracts of the leaves of three Piper species (Piper guineense Schum and Thonn., Piper nigrum L. and Piper umbellatum L.) (Piperaceae) for their polyphenolic concentration and free radical scavenging activity. The Folin-Ciocalteu method was used to assess the polyphenolic concentration while the reducing power, 1,1-diphenyl-2-picrylhydrazyl (DPPH), superoxide radical, hydroxyl radical, nitric oxide scavenging and metal chelating activities were employed in studying the radical scavenging activity of the extracts. All three spices exhibited a marked polyphenolic concentration and dose dependent free radical scavenging activity. The free polyphenolic concentration of the three spices was in the order P. umbellatum (15.9 +/- 1.9 mg/g) > P. guineense (12.6 +/- 0.3 mg/g) > P. nigrum (9.8 +/- 0.8). The three Piper extracts exhibited a 79.8-89.9% scavenging effect on DPPH, an 85.1-97.9% scavenging effect on nitric oxide at a dose level of 10 mg/ml and a 47.1-51.6% scavenging effect on superoxide radical at a dose level of 8 mg/ml extraction. Piper extracts also exhibited a 57-76.1% scavenging effect on hydroxyl radical at 5 mg/ml, a 0.4-0.6 reducing power and an 88.3-93.9% metal chelating activity at a dose level of 8 mg/ml of extract. Thus, these Piper species can play a role in the modulation of free radical induced disorders.     

Inhibition of H1N1 influenza A virus growth and induction of inflammatory mediators by the isoquinoline alkaloid berberine and extracts of goldenseal (Hydrastis canadensis)

In this study we tested whether the isoquinoline alkaloid berberine can inhibit the growth of influenza A. Our experiments showed strong inhibition of the growth of H1N1 influenza A strains PR/8/34 or WS/33 in RAW 264.7 macrophage-like cells, A549 human lung epithelial-derived cells and murine bone marrow derived macrophages, but not MDCK canine kidney cells. Studies of the mechanism underlying this effect suggest that berberine acts post-translationally to inhibit virus protein trafficking/maturation which in turn inhibits virus growth. Berberine was also evaluated for its ability to inhibit production of TNF-α and PGE(2) from A/PR/8/34 infected-RAW 264.7 cells. Our studies revealed strong inhibition of production of both mediators and suggest that this effect is distinct from the anti-viral effect. Finally, we asked whether berberine-containing ethanol extracts of goldenseal also inhibit the growth of influenza A and production of inflammatory mediators. We found strong effectiveness at high concentrations, although upon dilution extracts were somewhat less effective than purified berberine. Taken together, our results suggest that berberine may indeed be useful for the treatment of infections with influenza A.     

In vitro hepatotoxicity of the cyanobacterial alkaloid cylindrospermopsin and related synthetic analogues.

Cylindrospermopsin (CY), a sulfate ester of a tricyclic guanidine substituted with a hydroxymethyluracil, is a cyanobacterial toxin of increasing environmental import as it frequently occurs in drinking water reservoirs. As a toxin, CY mainly targets the liver but also involves other organs. In hepatocytes CY inhibits the synthesis of protein and of glutathione, leading to cell death. The total chemical synthesis of CY has recently been reported (Xie et al., 2000, J. Am. Chem. Soc. 22, 5017-5024). The synthesis has provided analogues of CY to study aspects of the relationship between chemical structure and activity that contribute to toxicity. Protein synthesis inhibition was measured in vitro using a rabbit reticulocyte system. Primary cultures of rat hepatocytes were used to determine the biological activity of CY and analogues in intact cells. Protein synthesis and cell glutathione levels were measured. We could distinguish between CY transport and biological activity by comparing the results in vitro to those in intact cells. The role of the sulfate group in CY toxicity was examined by comparing biological effects of CY with that of CY-DIOL (synthetic CY lacking the sulfate group). The sulfate group was found not to play a role in CY activity or in its uptake into cells, since there was no significant difference in biological activity in vitro or in cells between natural CY and CY-DIOL. The orientation of the hydroxyl group at C7 also had no impact on biological activity or transport of CY, since the C7 epimer of CY (EPI-CY) and the corresponding diol (EPI-DIOL) had activity similar to RAC-CY in vitro and in intact cells. AB-MODEL, the analogue lacking an intact C ring, and the methyl and hydroxyl groups of ring A could inhibit protein synthesis (but at concentrations 500-1000-fold higher than natural CY). Other structurally simpler synthetic analogues lacked biological activity.     

In vitro studies on metabolism of salvinorin A

Microbial transformation of natural products is a well established model for mammalian metabolism. Salvinorin A, a diterpenoid isolated from the hallucinogenic mint Salvia divinorum Epling & Játiva-M (Lamiaceae), is a potent non-nitrogenous κ-opioid receptor agonist. The metabolism of salvinorin A has still not yet been well established. Thirty fungal species were screened for the ability to metabolize salvinorin A. We observed that salvinorin A undergoes fast hydrolysis of the acetate group at carbon atom C2, resulting in formation of the pharmacologically inactive product, salvinorin B. Ex vivo experiments were also performed using organelle fractions isolated from rat liver and brain. Crude tissue homogenate and individual organelles show that the primary route of salvinorin A metabolism is hydrolysis to salvinorin B. No metabolic transformation of salvinorin B was observed in these studies.     

In vitro studies on the microsomal N-oxidation of N-substituted benzamidines

Studies of the Microsomal N-Oxidation of N-Substituted Benzamidines in Vitro The metabolism of the N-mono- and N,N-disubstituted benzamidines 4 and 7 was studied employing 9000 g supernatant fractions of rabbit liver homogenates as enzyme sources. The potential metabolites, i. e. the amidoximes 5 and 8 and the N-hydroxyamidine 6b , were synthesized and used as reference products. Among the N-substituted derivatives only N-phenylbenzamidine (4a) is metabolized with detectable yields (t.l.c./m.s.) to the amidoxime 5a. Considering this finding and the metabolism of other nitrogen-containing functional groups, it is assumed that the cytochrome P-450 system preferentially oxidizes NH₂ groups.