Anticholinesterse and antioxidant investigations of crude extracts, subsequent fractions, saponins and flavonoids of atriplex laciniata L: potential effectiveness in Alzheimer's and other neurological disorders

BACKGROUND: Atriplex laciniata L. was investigated for phenolic, flavonoid contents, antioxidant, anticholinesterase activities, in an attempt to explore its effectiveness in Alzheimer's and other neurological disorders. Plant crude methanolic extract (Al.MeF), subsequent fractions; n-hexane (Al.HxF), chloroform (Al.CfF), ethyl acetate (Al.EaF), aqueous (Al.WtF), Saponins (Al.SPF) and Flavonoids (Al.FLVF) were investigated for DPPH, ABTS and H2O2 free radical scavenging activities. Further these extracts were subjected to acetylcholinesterase (AChE) & butyrylcholinesterase (BChE) inhibitory activities using Ellman's assay. Phenolic and Flavonoid contents were determined and expressed in mg Gallic acid GAE/g and Rutin RTE/g of samples respectively. RESULTS: In DPPH free radicals scavenging assay, Al.FLVF, Al.SPF and Al.MeF showed highest activity causing 89.41 ± 0.55, 83.37 ± 0.34 and 83.37 ± 0.34% inhibition of free radicals respectively at 1 mg/mL concentration. IC50 for these fractions were 33, 83 and 82 µg/mL respectively. Similarly, plant extracts showed high ABTS scavenging potential, i.e. Al.FLVF (90.34 ± 0.55), Al.CfF (83.42 ± 0.57), Al.MeF (81.49 ± 0.60) with IC50 of 30, 190 and 70 µg/ml respectively. further, H2O2 percent scavenging was highly appraised in Al.FLVF (91.29 ±0.53, IC50 75), Al.SPF (85.35 ±0.61, IC50 70) and Al.EaF (83.48 ± 0.67, IC50 270 µg/mL). All fractions exhibited concentration dependent AChE inhibitory activity as; Al.FLVF, 88.31 ± 0.57 (IC50 70 µg/mL), Al.SPF, 84.36 ± 0.64 (IC50 90 µg/mL), Al.MeF, 78.65 ± 0.70 (IC50 280 µg/mL), Al.EaF, 77.45 ± 0.46 (IC50 270 µg/mL) and Al.WtF 72.44 ± 0.58 (IC50 263 µg/mL) at 1 mg/mL. Likewise the percent BChE inhibitory activity was most obvious in Al.FLVF 85.46 ± 0.62 (IC50 100 µg/mL), Al.CfF 83.49 ± 0.46 (IC50 160 µg/mL), Al.MeF 82.68 ± 0.60 (IC50 220 µg/mL) and Al.SPF 80.37 ± 0.54 (IC50 120 µg/mL). CONCLUSIONS: These results stipulate that A. laciniata is enriched with phenolic and flavonoid contents that possess significant antioxidant and anticholinestrase effects. This provide pharmacological basis for the presence of compounds that may be effective in Alzheimer's and other neurological disorders.

Leishmanicidal and cholinesterase inhibiting activities of phenolic compounds of Dimorphandra gardneriana and Platymiscium floribundum, native plants from Caatinga biome / Atividade inibitória Leishmanicida e Colinesterásica de Compostos Fenólicos de Dimorphandra gardneriana e Platymiscium fliribunbum, plantas nativas do bioma Caatinga

In recent years, the Brazilian Health Ministry and the World Health Organization have supported research into new technologies that may contribute to the surveillance, new treatments, and control of visceral leishmaniasis within the country. In light of this, the aim of this study was to isolate compounds from plants of the Caatinga biome, and to investigate their toxicity against promastigote and amastigote forms of Leishmania infantum chagasi, the main responsible parasite for South American visceral leishmaniasis, and evaluate their ability to inhibit acetylcholinesterase enzyme (AChE). A screen assay using luciferase-expressing promastigote form and an in situ ELISA assay were used to measure the viability of promastigote and amastigote forms, respectively, after exposure to these substances. The MTT colorimetric assay was performed to determine the toxicity of these compounds in murine monocytic RAW 264.7 cell line. All compounds were tested in vitro for their anti-cholinesterase properties. A coumarin, scoparone, was isolated from Platymiscium floribundum stems, and the flavonoids rutin and quercetin were isolated from Dimorphandra gardneriana beans. These compounds were purified using silica gel column chromatography, eluted with organic solvents in mixtures of increasing polarity, and identified by spectral analysis. In the leishmanicidal assays, the compounds showed dose-dependent efficacy against the extracellular promastigote forms, with an EC50 for scoporone of 21.4µg/mL, quercetin and rutin 26 and 30.3µg/mL, respectively. The flavonoids presented comparable results to the positive control drug, amphotericin B, against the amastigote forms with EC50 for quercetin and rutin of 10.6 and 43.3µg/mL, respectively. All compounds inhibited AChE with inhibition zones varying from 0.8 to 0.6, indicating a possible mechanism of action for leishmacicidal activity.

Nos últimos anos, o Ministério da Saúde do Brasil e a Organização Mundial da Saúde tem apoiado a investigação de novas tecnologias que possam contribuir para a vigilância, novos tratamentos e controle da leishmaniose visceral no país. Assim, o objetivo deste trabalho foi isolar compostos de plantas do bioma Caatinga, e investigar a toxicidade destes compostos contra as formas promastigotas e amastigotas de Leishmania infantum chagasi, principal parasita responsável pela leishmaniose visceral na América do Sul, e avaliar a sua capacidade para inibir a enzima acetil-colinesterase (AChE). Após a exposição aos compostos em estudo, foram realizados testes utilizando a forma promastigota que expressa luciferase e ELISA in situ para medir a viabilidade das formas promastigotas e amastigota, respectivamente. O ensaio colorimétrico MTT foi realizado para determinar a toxicidade destas substâncias utilizando células monocíticas murina RAW 264.7. Todos os compostos foram testados in vitro para as sua propriedade anti-colinesterásica. Um cumarina, escoparona, foi isolada a partir de hastes de Platymiscium floribundum, e os flavonóides, rutina e quercetina, foram isolados a partir de grãos de Dimorphandra gardneriana. Estes compostos foram purificados, utilizando cromatografia em coluna gel eluída com solventes orgânicos em misturas de polaridade crescente, e identificados por análise espectral. Nos ensaios leishmanicidas, os compostos fenólicos mostraram eficácia contra as formas extracelulares promastigotas, com EC50 para escoporona de 21.4µg/mL e para quercetina e rutina 26 e 30.3µg/mL, respectivamente. Os flavonóides apresentaram resultados comparáveis à droga controle, a anfotericina B, contra as formas amastigotas com EC50 para quercetina e rutina de 10.6 e 43.3µg/mL, respectivamente. Os compostos inibiram a enzima AChE com halos de inibição variando de 0,8 a 0,6cm, indicando um possível mecanismo de ação para a atividade leishmanicida.

Acetylcholinesterase inhibition starting from extracts of Bauhinia variegata L., Bauhinia var. candida (Aiton) Buch.-Ham., and Bauhinia ungulata L / Inibição de acetilcolinesterase a partir de extratos de Bauhinia variegata L., Bauhinia variegata var. candida (Aiton) Buch.-Ham., e Bauhinia ungulata L

INTRODUCTION: A treatment to the Alzheimer's disease consists inhibition of the acetylcholinesterase, which is responsible for the acetylcholine control in the synapses. METHODS: We have investigated the potential of inhibition of the acetylcholinesterase produced by hexane extracts of leaves, branches, and flowers from three Bauhinia specimens, which is based on the technique of thin layer chromatography and on identifying the organ of the plant that possesses larger concentration of inhibitors. RESULTS: Retention factor analysis shows values of 0.31aA, 0.31aA, and 0.46aB for flowers B. variegata, B. var. candida, and B. ungulata, respectively. CONCLUSIONS: The flower extract of B. ungulata is the most suitable for further studies on this inhibition.

INTRODUÇÃO: Um tratamento para a doença de Alzheimer consiste na inibição da acetilcolinesterase, responsável pelo controle de acetilcolina nas sinapses. MÉTODOS: Nós investigamos o potencial de inibição da acetilcolinesterase produzido por extratos hexânicos de folhas, ramos e flores de três espécimes de Bauhinia, realizado a partir da cromatografia em camada delgada e que órgão possui maior concentração de inibidores. RESULTADOS: Os fatores de retenção apresentaram 0,31aA, 0,31aA e 0,46aB para as flores de B. variegata, B. var. candida e B. ungulata respectivamente. CONCLUSÕES: O extrato de flores de B. ungulata é o mais satisfatório para futuros estudos desta inibição.

Acetylcholinesterase inhibition by somes promising Brazilian medicinal plants / Plantas medicinais brasileiras promissoras para inibição da acetilcolinesterase

A microplate assay and a thin-layer chromatography (TLC) "in situ" assay based on the Ellman assay was used to screen for acetylcholinesterase inhibitors from ethyl acetate and methanol extracts of Brazilian medicinal plants of families that, according to the literature, have traditional uses that might be connected with acetylcholinesterase inhibition. Eighteen species belonging to Convolvulaceae, Crassulaceae, Euphorbiaceae, Leguminosae, Malvaceae, Moraceae, Nyctaginaceae and Rutaceae families were tested. The most active plants were Ipomoea asarifolia (IC50 = 0.12 mg/mL), Jatropha curcas (IC50 = 0.25 mg/mL), Jatropha gossypiifolia (IC50 = 0.05 mg/mL), Kalanchoe brasiliensis (IC50 = 0.16 mg/mL) and Senna alata (IC50 = 0.08 mg/mL). The most promising extracts were the Jatropha gossypiifolia and Senna alata species assuming there were compounds with a similar activity to galanthamine, which should contain about 1 percent of an active compound, or if present at lower levels even more active compounds than galanthamine (IC50 = 0.37 x 10-3 mg/mL) should be present.

Os ensaios de microplaca e cromatografia em camada delgada com base no ensaio de Ellman foram usados para triagem de inibidores da acetilcolinesterase dos extratos acetato de etila e metanol de plantas medicinais brasileiras de famílias que, segundo a literatura, tem usos tradicionais que podem estar relacionadas com a inibição da acetilcolinesterase, enzima associada ao mal de Alzheimer. Dezoito plantas das famílias: Convolvulaceae, Crassulaceae, Euphorbiaceae, Leguminosae, Malvaceae, Moraceae, Nyctaginaceae e Rutaceae foram testadas. As espécies mais ativas foram Ipomoea asarifolia (CI₅₀ = 0,12 mg/mL), Jatropha curcas (CI₅₀ = 0,25 mg/mL), Jatropha gossypiifolia (CI₅₀ = 0,05 mg/mL), Kalanchoe brasiliensis (CI₅₀ = 0,16 mg/mL) e Senna alata (CI₅₀ = 0,08 mg/mL). Os extratos mais promissores foram os das espécies Jatropha gossypiifolia e Senna alata, assumindo a presença de compostos com atividade semelhante à galantamina que deve conter cerca de 1 por cento de um composto ativo, ou se presentes em menores níveis ainda mais compostos ativos que a galantamina (CI₅₀ = 0,37 x10^–3 mg/mL) devem estar presentes.

Two fast screening methods (GC-MS and TLC-ChEI assay) for rapid evaluation of potential anticholinesterasic indole alkaloids in complex mixtures

The pharmacotherapyfor Alzheimer's disease (AD) includes the use of acetylcholinesterase inhibitors (AChEI). Recent investigations for novel AD therapeutic agents from plants suggested that Tabernaemontana genus is a promising source of novel anticholinesterasic indole alkaloids. In this work two fast screening techniques were combined in order to easily identify novel cholinesterase inhibitors (ChEI). Gas chromatography-mass spectrometry (GC-MS) of the less polar alkaloidic fractions obtained from the acid-base extraction of the stalk of T. laeta revealed thirteen monoindole alkaloids, four of them confirmed by co-injection with previously isolated alkaloids. The others were tentatively identified by mass fragmentation analysis. By gas chromatography with flame ionization detection (GC-FID) and using isatin as internal standard, affinisine and voachalotine were determined as major compounds. These fractions and fourteen previously isolated alkaloids, obtained from root bark of T. laeta and T. hystrix were investigated for acetyl (AChE) and butyrylcholinesterase (BuChE) inhibitory activities by the modified Ellman's method in thin layer chromatography(TLC-ChEI). Results showed selective inhibition of the alkaloids heyneanine and Nb-methylvoachalotine for BuChE, and 19-epi-isovoacristine for AChE, whereas olivacine, affinisine, ibogamine, affinine, conodurine and hystrixnineinhibited both enzymes. In addition to confirming that monoterpenoid indole alkaloids can be novel therapeutic agents for AD, this is the first report of the ChEI activity of olivacine, a pyridocarbazole alkaloid.

Dentre os tratamentos da doença de Alzheimer (DA) está o uso de inibidores da enzima acetilcolinesterase. Pesquisas recentes visando a descoberta de novos agentes terapêuticos naturais para esta doença sugerem que o gênero Tabernaemontana é uma fonte promissora de alcalóides indólicos anticolinesterásicos. Neste trabalho, duas técnicas de análise em mistura foram associadas de modo a identificar facilmente novos inibidores colinesterásicos. A cromatografia em fase gasosa acoplada a espectrometria de massas (CG-EM) das frações alcaloídicas apolares, obtidas da extração ácido-base do caule de T. laeta, revelou a presença de treze alcalóides monoindólicos, quatro deles confirmados por co-injeção com padrões previamente isolados. Os outros alcalóides foram tentativamente identificados pelo padrão de fragmentação de massas. Por cromatografia em fase gasosa com detecção por ionização de chama (CG-DIC) e utilizando isatina como padrão interno, affinisina e voachalotina foram identificadas como substâncias majoritárias. As frações alcaloídicas obtidas e os quatorze alcalóides previamente isolados das raízes de T. laeta e T. hystrix foram analisados quanto à atividade inibitória das enzimas acetil (AChE) e butirilcolinesterase (BuChE) pelo método de Ellman em cromatografia em camada delgada (CCD-ChEI). Os resultados revelaram uma inibição seletiva dos alcalóides heyneanina e Nb-metilvoachalotina para BuChE e de 19-epi-isovoacristina para AChE, enquanto que olivacina, affinisina, ibogamina, affinina, conodurina e hystrixnina inibiram ambas as enzimas. Além de confirmar que alcalóides indólicos monoterpênicos são agentes terapêuticos promissores para o tratamento da DA, este é o primeiro relato da atividade anticolinesterásica de olivacina, um alcalóide piridocarbazólico.

In vitro inhibition of acetylcholinesterase by crude plant extracts from Colombian flora

The methanol extracts from five different plant families (Asteraceae, Euphorbiaceae, Melastomataceae, Rubiaceae, and Solanaceae) collected at Regional Natural Park Ucumarí (Colombia), were screened for their acetylcholinesterase inhibitory activity through the modified Ellman's spectrophotometric method. The best inhibitory activities on this study were shown by the extracts of Solanum leucocarpum Dunal (IC50 = 204.59 mg/l) and Witheringia coccoloboides (Damm) (IC50 = 220.68 mg/l), both plants belonging to the Solanaceae family.