Mutagenicity of pyrrolizidine alkaloids in the Salmonella/mammalian-microsome test.

The mutagenicities of 7 pyrrolizidine alkaloids to Salmonella typhimurium TA100 were demonstrated by a modified Ames's method. The pyrrolizidine alkaloids found to be mutagenic were clivorine, fukinotoxin, heliotrine, lasiocarpine, ligularidine, LX201 and senkirkine. Pre-incubation of these alkaloids with S9 mix and bacteria in a liquid medium was essential for demonstration of their mutagenicities.     

Further Daphniphyllum Alkaloids with Insecticidal Activity from the Bark of Daphniphyllum macropodum Miq.

Two new Daphniphyllum alkaloids, macropodumines J and K ( 1 and 2 , resp.), together with six known structurally related alkaloids, 3 – 8 , were isolated from the bark of Daphniphyllum macropodum Miq . The structures of the new compounds 1 and 2 were elucidated on the basis of a comprehensive analysis of their spectroscopic and chemical data. Macropodumine J ( 1 ) contains a CN group which is relatively rare in naturally occurring alkaloids. All isolated compounds were tested for their insecticidal activities against a number of insect species. Daphtenidine C ( 5 ) is the most active compound against Plutella xylostella. This is the first report of insecticidal properties of Daphniphyllum alkaloids.     

The effect of varying alkaloid concentrations on the feeding behavior of gypsy moth larvae, Lymantria dispar (L.) (Lepidoptera: Lymantriidae)

Nine alkaloids (acridine, aristolochic acid, atropine, berberine, caffeine, nicotine, scopolamine, sparteine, and strychnine) were evaluated as feeding deterrents for gypsy moth larvae (Lymantria dispar (L.); Lepidoptera: Lymantriidae). Our aim was to determine and compare the taste threshold concentrations, as well as the ED₅₀ values, of the nine alkaloids to determine their potency as feeding deterrents. The alkaloids were applied to disks cut from red oak leaves (Quercus rubra) (L.), a plant species highly favored by larvae of this polyphagous insect species. We used two-choice feeding bioassays to test a broad range of biologically relevant alkaloid concentrations spanning five logarithmic steps. We observed increasing feeding deterrent responses for all the alkaloids tested and found that the alkaloids tested exhibited different deterrency threshold concentrations ranging from 0.1 to 10 mM. In conclusion, it appears that this generalist insect species bears a relatively high sensitivity to these alkaloids, which confirms behavioral observations that it avoids foliage containing alkaloids. Berberine and aristolochic acid were found to have the lowest ED₅₀ values and were the most potent antifeedants. Handling Editor: Joseph Dickens.     

The metabolic activation of 4-nitro-o-phenylenediamine by chlorophyll-containing plant extracts: The relationship between mutagenicity and antimutagenicity

Chlorophyllin, the sodium and copper salt of chloropyll, and chlorophyll a, and chlorophyll b were tested for their ability to inhibit the mutagenic activity of the direct-acting mutagen 4-nitro-o-phenylenediamine (NOP) and its plant-activated mutagenic enhancement. All three forms of chlorophyll were antimutagenic against both NOP and its plant-activated product, with chlorophyllin proving most effective. Chlorophyll-containing plant extracts, however, proved very efficient at activating NOP into a mutagen of greater potency. When these extracts were assayed for total chlorophyll content it was found that they contained far less chlorophyll than was required for an antimutagenic effect to occur. Thus, the balance between chemical mutagen activation and/or enhancement by chlorophyll-containing plant extracts and the potential antimutagenicity of these plant extracts is a function of chlorophyll concentration. The data presented here indicate that this balance must be taken into consideration in future studies investigating the efficacy of complex natural plant extracts as antimutagenic substances.     

Chemistry and in vitro mutagenicity of 2-aminofluorene derivatives

A study on the relationship between mutagenic activity and chemical reactivity of a series of 2-fluorenylamino and hydroxylamino derivatives has been carried out by assaying their ability to revert the Salmonella typhimurium strain TA98. The mutagenic potency of the fluorenamides increased with increasing availability of the amidic hydrogen for abstraction and tertiary amides were quite inactive. N-Hydroxy and N-acyloxy derivatives were directly mutagenic and increased their mutagenic activity after metabolic conversion by liver S9. N-Hydroxy-2-benzoylaminofluorene, inactive without S9, after activation was the most mutagenic. Of a pair of N-acyloxy-derivatives, N-benzoyloxy-2-acetylaminofluorene, which undergoes rearrangement of the benzoyloxy group from nitrogen to ring carbons even at room temperature, was less potent than N-acetyloxy-2-acetylamino-fluorene whose rearrangement occurs at higher temperatures. Corresponding C-1 and C-3 benzoyloxy and acetyloxy derivatives were found ineffective in this assay in agreement with previous reports on the hydroxy series. N-Chloro-2-amino-(or acetylamino)fluorene were found more active than the corresponding N-hydroxy analogs in the presence of S9, thus suggesting an alternate pathway for activation, likely a direct conversion to electrophilic species. Furthermore, in contrast with inactivity of ring hydroxy and acyloxy derivatives, 3-chloro-2-acetylaminofluorene retained mutagenic activity. Finally 2,2'-azoxyfluorene, the ultimated oxidation product of N-hydroxyaminofluorene, tested in vitro and in vivo experiments, was found completely inactive.     

Isolation and Antimacrofouling Activity of Indole and Furoquinoline Alkaloids from ‘Guatambú’ Trees (Aspidosperma australe and Balfourodendron riedelianum)

In this work, the antifouling activity of five alkaloids, isolated from trees of the Atlantic rainforest, was studied. The tested alkaloids were olivacine ( 1 ), uleine ( 2 ) and N‐methyltetrahydroellipticine ( 3 ) from Aspidosperma australe (‘yellow guatambú’) and the furoquinoline alkaloids kokusaginine ( 4 ) and flindersiamine ( 5 ) from Balfourodendron riedelianum (‘white guatambú’). All these compounds can be isolated from their natural sources in high yields in a sustainable way. The five compounds were subjected to laboratory tests (attachment test of the mussel Mytilus edulis platensis) and field trials, by incorporation into soluble matrix paints, and 45 days of exposure of the painted panels in the sea. The results show that compound 3 is a very potent antifoulant, and that compounds 4 and 5 are also very active, while compounds 1 and 2 did not show any significant antifouling activity. These results open the way for the development of environmentally friendly antifouling agents, based on abundant and easy‐to‐purify compounds that can be obtained in a sustainable way.     

Uptake and metabolism of pyrrolizidine alkaloids in<Emphasis Type="Italic"> Longitarsus</Emphasis> flea beetles (Coleoptera: Chrysomelidae) adapted and non-adapted to alkaloid-containing host plants

Several Longitarsus flea beetle species sequester pyrrolizidine alkaloids acquired from their Asteraceae and Boraginaceae host plants. We carried out feeding and injection experiments using radioactively labeled pyrrolizidine alkaloids to investigate the physiological mechanisms of uptake, metabolism and storage of alkaloids in adult beetles. We examined six Longitarsus species belonging to different phylogenetic clades in a comparative approach. All species that accepted pyrrolizidine alkaloids in a preceding food choice study showed the ability both to store pyrrolizidine alkaloid N-oxides and to metabolize tertiary pyrrolizidine alkaloids into their N-oxides. Regardless of whether the beetles' natural host plants contain pyrrolizidine alkaloids or not, these species were found to possess an oxidizing enzyme. This oxygenase appears to be specific to pyrrolizidine alkaloids: [³H]Atropine and [¹⁴C]nicotine, two alkaloids not related to pyrrolizidine alkaloids, were neither stored nor N-oxidized by any of the tested species. One species, L. australis, that strictly avoids pyrrolizidine alkaloids behaviorally, exhibited a lack of adaptations to pyrrolizidine alkaloids on a physiological level as well. After injection of tertiary [¹⁴C]senecionine, beetles of this species neither N-oxidized nor stored the compounds, in contrast to L. jacobaeae, an adapted species that underwent the same treatment. L. jacobaeae demonstrated the same efficiency in N-oxidation and storage when fed or injected with tertiary [¹⁴C]senecionine.Communicated by G. Heldmaier     

Isoquinoline alkaloids from Mahonia aquifolium stem bark are active against Malassezia spp

Bioassay directed fractionation of crude extract fromMahonia aquifolium led to the isolation of fraction A (bisbenzylisoquinoline alkaloid complex, BBI) and a fraction of protoberberine alkaloids, where the major compounds berberine and jatrorrhizine were isolated as their iodides. The antifungal activity of the crude extract, two protoberberine alkaloids and BBI fromM. aquifolium stem bark were evaluated against six strains ofMalassezia spp. The compounds tested were generally found to possess only weak to moderate antifungal properties: the MICs for individual strains were in the range ≤50–≥1000 mg/L.     

Mutagenicity of pyrrolizidine alkaloids in the Salmonella typhimurium/mammalian microsome system

The mutagenicity of a series of pyrrolizidine alkaloids, and of extracts from several Italian Senecio species containing pyrrolizidine alkaloids, including S. inaequidens, S. fuchsii and S. cacaliaster, were tested using the Salmonella typhimurium/mammalian microsome system. Retrorsine, senecivernine, seneciphylline and the Senecio extracts showed a weakly mutagenic activity.     

Effect of various alkyl and unsaturated substituents on the mutagenicity of some nitrophenyl thioethers.

A variety of nitro-substituted phenyl alkyl/aryl thioethers and nitroso-substituted phenyl alkyl/aryl thioethers have been synthesized and tested for their mutagenicity towards Salmonella typhimurium strain TA100, TA98, TA98NR and TA98/1,8-DNP(6) in the absence of S9 mix. The relative order of mutagenicity in TA98 and TA100 among p-nitrophenyl thioethers having alkyl or aryl substituents is allyl>phenyl>benzyl>butyl>propyl>ethyl>methyl. Compounds having an alkyl chain C(6) to C(12) were found to be non-mutagenic. Among the various positional isomers (ortho, meta and para) of nitro-substituted diphenyl thioethers only the compounds having the -NO(2) function at the para position is mutagenic, whereas compounds having a -NO(2) function at ortho and meta are non-mutagenic. However, the reduced intermediate, ortho-nitroso derivative was found to be mutagenic in all the four strains but the meta-nitroso derivative was found to be non-mutagenic. All mutagens were found to be non-mutagenic when tested in nitroreductase deficient strain TA98NR, whereas their nitroso intermediates are found to be mutagenic. A substantial fall in the mutagenic activity is observed when some mutagens are tested in O-acetyltransferase deficient strain TA98/1,8-DNP(6).     

Mutagenic activities of ten imidazole derivatives in <Emphasis Type="Italic">Salmonella typhimurium</Emphasis>

Ten imidazole derivatives were tested for mutagenicity in Salmonella typhimurium strains TA98 and TA100 both in the absence and presence of metabolic activation by the microsomal fraction S9 mix. In a general manner, derivatives tested exhibited a greater mutagenic activity in the TA100 strain comparing to the responses in TA 98. In the standard plate incorporation assay, 8 of these substances (80%) were found to be mutagenic for at least one of the two strains in the presence or absence of metabolic activation. Two compounds showed positive results in TA98 and 6 compounds were also mutagenic in TA100 without S9. In the presence of S9 mix, all of the 10 substances were non-mutagenic in TA98, whereas 4 compounds were positive in TA100. The results suggested the mutagenic potentials of the imidazole derivatives particularly inducing the reversion of base-pair substitutions. According to the structure-activity relationships phenyl groups in position 2 with different substituents can confer the mutagenic activity of the tested compounds. Methyl groups in different positions of these phenyl substituents can cause different types of mutations. This mutagenic effect is observed more clearly when the phenyl group is inhibited with a nitro group.     

Low antiplasmodial activity of alkaloids and amides from the stem bark of Zanthoxylum rubescens (Rutaceae)

The stem bark of Zanthoxylum rubescens (syn. Fagara rubescens) is used for treating fevers associated with malaria in the Ivory Coast. Three alkaloids: N-nornitidine, 7,9-dimethoxy-2,3-methylenedioxybenzophenanthridine, and bis[6-5,6-dihydrochelerythrinyl)] ether; and two amides: zanthomamide and lemairamide, were isolated from the stem bark of this plant. These compounds were screened in vitro against the chloroquine-sensitive 3D7 strain and the chloroquine-resistant FCM29 strain of P. falciparum. N-nornitidine was found to be inactive. 7,9-dimethoxy-2,3-methylenedioxybenzophenanthridine, lemairamide and zanthomamide showed weak activity with average IC50 values ranging from 45.6 microM to 149.9 microM. Bis[6-15,6-dihydrochelerythrinyl)] ether was the most active of the tested compounds with mean IC50s of 14.9 +/- 1.4 microM in FCM29 strain and 15.3 +/- 3.4 microM in 3D7 strain (approximately 58 to approximately 1130 times less active than chloroquine respectively). The anti-Plasmodium activities of the tested alkaloids of Z. rubescens were low; and do not encourage the use of this plant as antimalarial.     

Mutagenicity of pyrrolizidine alkaloids in the Salmonella typhimurium/mammalian microsome system.

The mutagenicity of a series of pyrrolizidine alkaloids, and of extracts from several Italian Senecio species containing pyrrolizidine alkaloids, including S. inaequidens, S. fuchsii and S. cacaliaster, were tested using the Salmonella typhimurium/mammalian microsome system. Retrorsine, senecivernine, seneciphylline and the Senecio extracts showed a weakly mutagenic activity.     

Mutagenicity of K-region derivatives of 1-nitropyrene; remarkable activity of 1- and 3-nitro-5H-phenanthro[4,5-bcd]pyran-5-one

The mutagenic activities toward S. typhimurium strains TA98 and TA100 of K-region derivatives of 1-nitropyrene and pyrene were determined. The compounds tested were trans-4,5-dihydro-4,5-dihydroxy-1-nitropyrene (Compound 3), trans-4,5-dihydro-4,5-dihydroxypyrene (Compound 4), 1-nitropyrene-4,5-quinone (Compound 5), 1-nitropyrene-9,10-quinone (Compound 6), pyrene-4,5-quinone (Compound 7), and the lactones, 1-nitro-5H-phenanthro[4,5-bcd]pyran-5-one (Compound 8), 3-nitro-5H-phenanthro[4,5-bcd]pyran-5-one (Compound 9), and 5H-phenanthro[4,5-bcd]pyran-5-one (Compound 10). Neither pyrene nor any of its K-region derivatives was mutagenic, either in the absence or presence of S9 mix at the doses tested. Of the K-region derivatives of 1-nitropyrene, the lactones (Compounds 8 and 9) were generally the most active; 0.25 microgram/plate induced 900-2200 revertants in TA98 or TA100 without activation. The 4,5-dihydrodiol (Compound 3), an established mammalian metabolite of 1-nitropyrene, was less mutagenic than was 1-nitropyrene in TA98, but was more mutagenic than was 1-nitropyrene in TA100, regardless of the presence of S9 mix. The quinones (Compounds 5 and 6) were less mutagenic than was 1-nitropyrene in the absence of S9 mix in both strains, but their activities were increased in the presence of S9 mix. The mutagenic activities of the lactones (Compounds 8 and 9) were lower in strains TA98NR and TA98/1,8-DNP6 than in TA98, indicating that nitro-reduction and esterification are involved in their activation. The results of this study indicate that K-region derivatives of 1-nitropyrene may be important in its metabolic activation.     

Structure-Activity Relationship of Benzophenanthridine Alkaloids from Zanthoxylum rhoifolium Having Antimicrobial Activity

Zanthoxylum rhoifolium (Rutaceae) is a plant alkaloid that grows in South America and has been used in Brazilian traditional medicine for the treatment of different health problems. The present study was designed to evaluate the antimicrobial activity of the steam bark crude methanol extract, fractions, and pure alkaloids of Z. rhoifolium. Its stem bark extracts exhibited a broad spectrum of antimicrobial activity, ranging from 12.5 to 100 µg/mL using bioautography method, and from 125 to 500 µg/mL in the microdilution bioassay. From the dichloromethane basic fraction, three furoquinoline alkaloids (1–3), and nine benzophenanthridine alkaloids (4–12) were isolated and the antimicrobial activity of the benzophenanthridine alkaloids is discussed in terms of structure-activity relationships. The alkaloid with the widest spectrum of activity was chelerythrine (10), followed by avicine (12) and dihydrochelerythrine (4). The minimal inhibitory concentrations of chelerythrine, of 1.50 µg/mL for all bacteria tested, and between 3.12 and 6.25 µg/mL for the yeast tested, show this compound to be a more powerful antimicrobial agent when compared with the other active alkaloids isolated from Z. rhoifolium. To verify the potential importance of the methylenedioxy group (ring A) of these alkaloids, chelerythrine was selected to represent the remainder of the benzophenanthridine alkaloids isolated in this work and was subjected to a demethylation reaction giving derivative 14. Compared to chelerythrine, the derivative (14) was less active against the tested bacteria and fungi. Kinetic measurements of the bacteriolytic activities of chelerythrine against the bacteria Bacillus subtilis (Gram-positive) and Escherichia coli (Gram-negative) were determined by optical density based on real time assay, suggesting that its mechanism of action is not bacteriolytic. The present study did not detect hemolytic effects of chelerythrine on erythrocytes and found a protective effect considering the decrease in TBARS and AOPP (advanced oxidized protein products) levels when compared to the control group.     

Purification of Debranching Enzyme from Mature Rice Seeds

Two indole alkaloids which induce the Epstein-Barr virus early antigen of Raji cells (B lymphocyte) were found in the cultured broth of Actinomycetes NA34-17, from which teleocidin B was also obtained. The active compounds isolated were identified from their spectral data and chemical evidence as (—)-indolactam V and (—)-14-O-acetyl indolactam V.     

Structure-activity relationships of a series of antitumoral 5,8-quinazolinediones in mutagenicity studies.

Four antitumoral 5,8-quinazolinediones were examined for their ability to induce mutation in Salmonella typhimurium. Each compound was tested at several concentrations in 4 strains. Relationships were established between the structure of the quinones and their mutagenic activities. The mutagenicity was influenced by (i) the nature of the substituent(s) of the quinonic moiety: the methoxyquinone had no mutagenic properties and the aziridinylquinones were mutagenic in the 4 strains with or without activation by S9 mix; (ii) the presence or the absence of a diaminopolymethylenic chain in the 4 position; (iii) the monomeric or the dimeric structure of the tested compound. Interestingly, the data indicated that the aziridinylquinazolinedione bearing the dimethylaminopropylamino chain in the 4 position was less mutagenic and had greater antitumor activity than the dimeric quinone.     

Microbial mutagenicity of 3- and 4-ring polycyclic aromatic sulfur heterocycles

The stable isomers of 3- and 4-ring polycyclic aromatic sulfur heterocycles were tested for mutagenicity in the Ames standard plate incorporation test and a liquid pre-incubation modification of the Ames test. Of the 4 three-ring compounds tested, only naphtho[1,2-b]thiophene was mutagenic. Of the four-ring compounds, 7 of 13 were mutagenic in the standard Ames or pre-incubation Ames test. The highest activity for the 4-ring compounds was observed for phenanthrol[3,4-b]thiophene, a compound of approximately the same mutagenic potency in the Ames test as benzo[a]pyrene. The other active 4-ring compounds were of considerable less mutagenic potency than phenanthrol[3,4-b]thiophene. Mutagenicity for two of the 4-ring aromatic thiophenes could only be detected in the liquid pre-incubation Ames test. Salmonella typhimurium TA100 was the most sensitive strain to mutagenesis by these compounds, followed by TA98. All mutagenesis was indirect, requiring metabolic activation.     

Profiling Auspicious Butyrylcholinesterase Inhibitory Activity of Two Herbal Molecules: Hyperforin and Hyuganin C

Cholinergic therapy based on cholinesterase (ChE) inhibitory drugs is the mainstay for the treatment of Alzheimer's disease. Therefore, an extensive research has been continuing for the discovery of drug candidates as inhibitors of acetyl‐ and butyrylcholinesterase. In this study, two natural molecules, e. g. hyperforin and hyuganin C were tested in vitro for their AChE and BChE inhibitory activity. Both of the compounds were ineffective against AChE, whereas hyperforin (IC₅₀=141.60±3.39 μm ) and hyuganin C (IC₅₀=38.86±1.69 μm ) were found to be the highly active inhibitors of BChE as compared to galantamine (IC₅₀=46.58±0.91 μm ) which was used as the reference. Then, these molecules were further proceeded to molecular docking experiments in order to establish their interactions at the active site of BChE. The molecular docking results indicated that both of them are able to block the access to key residues in the catalytic triad of the enzyme, while they complement some of the hydrophobic residues of the cavity, what is consistent with our in vitro data. While both compounds were predicted as mutagenic, only hyuganin C showed hepatotoxicity in in silico analysis. According to whole outcomes that we obtained, particularly hyuganin C besides hyperforin are the promising BChE inhibitors, which can be the promising compounds for AD therapy.     

Adaptation of the Salmonella/mammalian microsome test to the determination of the mutagenic properties of mineral oils

Two techniques allowing the determination of the mutagenicity of lipophilic compounds such as mineral oils with the Ames test have been developed by using benzo[a]pyrene (BP) dissolved in white oil as a synthetic reference oil. The first technique involves prior extraction of polynuclear aromatic hydrocarbons (PAH) with dimethyl sulfoxide. In the second method, which proved simpler and of more general use, the compounds to be tested are directly dispersed in aqueous medium with Tween 80. The use of these techniques made possible the study of mutagenicity of various kinds of mineral oil. Mutagenic activity was found in used crankcase oils, and also in petroleum distillates but much less in solvent-refined oils. A good correlation was observed between mutagenic activity and PAH content but not BP content of oils.Because of their peculiar response to the test, petroleum distillates were studied in more detail. When added in low amounts to pure PAH compounds such as BP, they enhanced its mutagenic activity (enhancement). When added in higher amounts, on the contrary, these oils completely inhibited BP mutagenic activity (inhibition effect). Both effects correlated well with the PAH content and the mutagenic activity of the petroleum distillates tested. These results explain the abnormal dose—response curves obtained with these petroleum distillates and the negative results regarding their mutagenic activity reported in earlier studies. A likely explanation is discussed for the enhancement and inhibition effects.