Interactions of Isoquinoline Alkaloids with Transition Metals Iron and Copper.

Data on alkaloid interactions with the physiologically important transition metals, iron and copper, are mostly lacking in the literature. However, these interactions can have important consequences in the treatment of both Alzheimer's disease and cancer. As isoquinoline alkaloids include galanthamine, an approved drug for Alzheimer's disease, as well as some potentially useful compounds with cytostatic potential, 28 members from this category of alkaloids were selected for a complex screening of interactions with iron and copper at four pathophysiologically relevant pH and in non-buffered conditions (dimethyl sulfoxide) by spectrophotometric methods in vitro. With the exception of the salts, all the alkaloids were able to chelate ferrous and ferric ions in non-buffered conditions, but only five of them (galanthine, glaucine, corydine, corydaline and tetrahydropalmatine) evoked some significant chelation at pH 7.5 and only the first two were also active at pH 6.8. By contrast, none of the tested alkaloids chelated cuprous or cupric ions. All the alkaloids, with the exception of the protopines, significantly reduced the ferric and cupric ions, with stronger effects on the latter. These effects were mostly dependent on the number of free aromatic hydroxyls, but not other hydroxyl groups. The most potent reductant was boldine. As most of the alkaloids chelated and reduced the ferric ions, additional experimental studies are needed to elucidate the biological relevance of these results, as chelation is expected to block reactive oxygen species formation, while reduction could have the opposite effect.

Semisynthetic Derivatives of Selected Amaryllidaceae Alkaloids as a New Class of Antimycobacterial Agents.

The search for novel antimycobacterial drugs is a matter of urgency, since tuberculosis is still one of the top ten causes of death from a single infectious agent, killing more than 1.4 million people worldwide each year. Nine Amaryllidaceae alkaloids (AAs) of various structural types have been screened for their antimycobacterial activity. Unfortunately, all were considered inactive, and thus a pilot series of aromatic esters of galanthamine, 3-O-methylpancracine, vittatine and maritidine were synthesized to increase biological activity. The semisynthetic derivatives of AAs were screened for their in vitro antimycobacterial activity against Mycobacterium tuberculosis H37Ra and two other mycobacterial strains (M. aurum, M. smegmatis) using a modified Microplate Alamar Blue Assay. The most active compounds were also studied for their in vitro hepatotoxicity on the hepatocellular carcinoma cell line HepG2. In general, the derivatization of the original AAs was associated with a significant increase in antimycobacterial activity. Several pilot derivatives were identified as compounds with micromolar MICs against M. tuberculosis H37Ra. Two derivatives of galanthamine, 1i and 1r, were selected for further structure optimalization to increase the selectivity index.

Bersavine: A Novel Bisbenzylisoquinoline Alkaloidwith Cytotoxic, Antiproliferative and Apoptosis-Inducing Effects on Human Leukemic Cells.

Bersavine is the new bisbenzylisoquinoline alkaloid isolated from the Berberis vulgaris L.(Berberidaceae) plant. The results of cytotoxicity screening 48 h post-treatment showed thatbersavine considerably inhibits the proliferation and viability of leukemic (Jurkat, MOLT-4), colon(HT-29), cervix (HeLa) and breast (MCF-7) cancer cells with IC50 values ranging from 8.1 to 11 µM.The viability and proliferation of leukemic Jurkat and MOLT-4 cells were decreased after bersavinetreatment in a time- and dose-dependent manner. Bersavine manifested concentration-dependentantiproliferative activity in human lung, breast, ovarian and hepatocellular carcinoma cell linesusing a xCELLigence assay. Significantly higher percentages of MOLT-4 cells exposed to bersavineat 20 µM for 24 h were arrested in the G1 phase of the cell cycle using the flow cytometry method.The higher percentage of apoptotic cells was measured after 24 h of bersavine treatment. Theupregulation of p53 phosphorylated on Ser392 was detected during the progression of MOLT-4 cellapoptosis. Mechanistically, bersavine-induced apoptosis is an effect of increased activity ofcaspases, while reduced proliferation seems dependent on increased Chk1 Ser345 phosphorylationand decreased Rb Ser807/811 phosphorylation in human leukemic cells.

Isoquinoline Alkaloids from Berberis vulgaris as Potential Lead Compounds for the Treatment of Alzheimer's Disease.

Three new alkaloids, bersavine (3), muraricine (4), and berbostrejdine (8), together with seven known isoquinoline alkaloids (1-2, 5-7, 9, and 10) were isolated from an alkaloidal extract of the root bark of Berberis vulgaris. The structures of the isolated compounds were determined by spectroscopic methods, including 1D and 2D NMR techniques, HRMS, and optical rotation, and by comparison of the obtained data with those in the literature. The NMR data of berbamine (5), aromoline (6), and obamegine (7) were completely assigned employing 2D NMR experiments. Alkaloids isolated in sufficient amounts were evaluated for their in vitro acetylcholinesterase, butyrylcholinesterase (BuChE), prolyl oligopeptidase, and glycogen synthase kinase-3ß inhibitory activities. Selected compounds were studied for their ability to permeate through the blood-brain barrier. Significant human BuChE ( hBuChE) inhibitory activity was demonstrated by 6 (IC50 = 0.82 ± 0.10 µM). The in vitro data were further supported by computational analysis that showed the accommodation of 6 in the active site of hBuChE.

In Vitro and In Silico Acetylcholinesterase Inhibitory Activity of Thalictricavine and Canadine and Their Predicted Penetration across the Blood-Brain Barrier.

In recent studies, several alkaloids acting as cholinesterase inhibitors were isolated from Corydalis cava (Papaveraceae). Inhibitory activities of (+)-thalictricavine (1) and (+)-canadine (2) on human acetylcholinesterase (hAChE) and butyrylcholinesterase (hBChE) were evaluated with the Ellman's spectrophotometric method. Molecular modeling was used to inspect the binding mode of compounds into the active site pocket of hAChE. The possible permeability of 1 and 2 through the blood⁻brain barrier (BBB) was predicted by the parallel artificial permeation assay (PAMPA) and logBB calculation. In vitro, 1 and 2 were found to be selective hAChE inhibitors with IC50 values of 0.38 ± 0.05 µM and 0.70 ± 0.07 µM, respectively, but against hBChE were considered inactive (IC50 values > 100 µM). Furthermore, both alkaloids demonstrated a competitive-type pattern of hAChE inhibition and bind, most probably, in the same AChE sub-site as its substrate. In silico docking experiments allowed us to confirm their binding poses into the active center of hAChE. Based on the PAMPA and logBB calculation, 2 is potentially centrally active, but for 1 BBB crossing is limited. In conclusion, 1 and 2 appear as potential lead compounds for the treatment of Alzheimer's disease.

Amaryllidaceae Alkaloids as Potential Glycogen Synthase Kinase-3ß Inhibitors.

Glycogen synthase kinase-3ß (GSK-3ß) is a multifunctional serine/threonine protein kinase that was originally identified as an enzyme involved in the control of glycogen metabolism. It plays a key role in diverse physiological processes including metabolism, the cell cycle, and gene expression by regulating a wide variety of well-known substances like glycogen synthase, tau-protein, and ß-catenin. Recent studies have identified GSK-3ß as a potential therapeutic target in Alzheimer´s disease, bipolar disorder, stroke, more than 15 types of cancer, and diabetes. GSK-3ß is one of the most attractive targets for medicinal chemists in the discovery, design, and synthesis of new selective potent inhibitors. In the current study, twenty-eight Amaryllidaceae alkaloids of various structural types were studied for their potency to inhibit GSK-3ß. Promising results have been demonstrated by alkaloids of the homolycorine-{9-O-demethylhomolycorine (IC50 = 30.00 ± 0.71 µM), masonine (IC50 = 27.81 ± 0.01 µM)}, and lycorine-types {caranine (IC50 = 30.75 ± 0.04 µM)}.

LC-MS/MS method for the determination of haemanthamine in rat plasma, bile and urine and its application to a pilot pharmacokinetic study.

Evidence gathered in various studies points to the fact that haemanthamine, an isoquinoline alkaloid, has multiple medicinally interesting characteristics, including antitumor, antileukemic, antioxidant, antiviral, anticonvulsant and antimalarial activity. This work presents, for the first time, a universal LC-MS/MS method for analysis of haemanthamine in plasma, bile and urine which has been verified in a pilot pharmacokinetic experiment on rats. Chromatographic separation was performed on a pentafluorophenyl core-shell column in gradient elution mode with a mobile phase consisting of acetonitrile-methanol-ammonium formate buffer. A sample preparation based on liquid-liquid extraction with methyl tert-butyl ether was employed with ambelline used as an internal standard. Quantification was performed using LC-MS-ESI(+) in Selected Reaction Monitoring mode. The method was validated according to the European Medicines Agency guideline in a concentration range of 0.1-10 µmol/L in plasma, bile and urine. The concentration-time profiles of haemanthamine in plasma, bile and urine after a single i.v. bolus of 10 mg/kg have been described for the first time. The presented study addresses the lack of information on haemanthamine pharmacokinetics and also introduces a new universal method of haemanthamine analysis in complex biological matrices. Copyright © 2015 John Wiley & Sons, Ltd.

Flavones Inhibit the Activity of AKR1B10, a Promising Therapeutic Target for Cancer Treatment.

AKR1B10 is an NADPH-dependent reductase that plays an important function in several physiological reactions such as the conversion of retinal to retinol, reduction of isoprenyl aldehydes, and biotransformation of procarcinogens and drugs. A growing body of evidence points to the important role of the enzyme in the development of several types of cancer (e.g., breast, hepatocellular), in which it is highly overexpressed. AKR1B10 is regarded as a therapeutic target for the treatment of these diseases, and potent and specific inhibitors may be promising therapeutic agents. Several inhibitors of AKR1B10 have been described, but the area of natural plant products has been investigated sparingly. In the present study almost 40 diverse phenolic compounds and alkaloids were examined for their ability to inhibit the recombinant AKR1B10 enzyme. The most potent inhibitors-apigenin, luteolin, and 7-hydroxyflavone-were further characterized in terms of IC50, selectivity, and mode of action. Molecular docking studies were also conducted, which identified putative binding residues important for the interaction. In addition, cellular studies demonstrated a significant inhibition of the AKR1B10-mediated reduction of daunorubicin in intact cells by these inhibitors without a considerable cytotoxic effect. Although these compounds are moderately potent and selective inhibitors of AKR1B10, they constitute a new structural type of AKR1B10 inhibitor and may serve as a template for the development of better inhibitors.

Comparison of an HPTLC method with the Reflectoquant assay for rapid determination of 5-hydroxymethylfurfural in honey.

5-Hydroxymethylfurfural (HMF) was analyzed in 17 botanical varieties of honey from 12 countries. A recently developed high-performance thin-layer chromatographic (HPTLC) method was limited because of increased matrix effects at higher honey sample loading. Therefore, the method was modified to achieve higher sensitivity and eliminate matrix interference by use of rectangular application combined with a focusing step. The HPTLC results were compared with results from the new spectrophotometric Reflectoquant hydroxymethylfurfural assay. Both methods had quantification limits of 4 mg kg(-1) and were suitable for rapid quantification of HMF in honey at the strictest regulated level of 15 mg kg(-1). Comparable results were obtained for the 17 honey samples, with a mean deviation of 2.9 mg kg(-1) (15%). The optimized HPTLC method was proved to be highly matrix-robust and was validated for the 17 different honey matrices (correlation coefficients ≥0.9994 (n = 6), mean intra-day precision 3.2% (n = 3 within a plate; n = 2 repeated within a day), mean inter-day precision 3.7% (n = 3), mean reproducibility over the whole procedure including sample preparation 4.1% (n = 2), and mean recovery 106.9% (n = 5 different concentrations; n = 4 different honey matrices). Recovery for a range of different application volumes, and thus for different honey matrix loading, differed by only ≤4.2%. HMF results when calculated by use of external calibration and by use of the standard addition method varied by 8.8%. Both revealed that any matrix effect was minor and that the original matrix interference problem was successfully solved.

Derivatives of Amaryllidaceae Alkaloid Ambelline as Selective Inhibitors of Hepatic Stage of Plasmodium berghei Infection In Vitro.

The incidence rate of malaria and the ensuing mortality prompts the development of novel antimalarial drugs. In this work, the activity of twenty-eight Amaryllidaceae alkaloids (1-28) belonging to seven different structural types was assessed, as well as twenty semisynthetic derivatives of the ß-crinane alkaloid ambelline (28a-28t) and eleven derivatives of the α-crinane alkaloid haemanthamine (29a-29k) against the hepatic stage of Plasmodium infection. Six of these derivatives (28h, 28m, 28n and 28r-28t) were newly synthesized and structurally identified. The most active compounds, 11-O-(3,5-dimethoxybenzoyl)ambelline (28m) and 11-O-(3,4,5-trimethoxybenzoyl)ambelline (28n), displayed IC50 values in the nanomolar range of 48 and 47 nM, respectively. Strikingly, the derivatives of haemanthamine (29) with analogous substituents did not display any significant activity, even though their structures are quite similar. Interestingly, all active derivatives were strictly selective against the hepatic stage of infection, as they did not demonstrate any activity against the blood stage of Plasmodium infection. As the hepatic stage is a bottleneck of the plasmodial infection, liver-selective compounds can be considered crucial for further development of the malaria prophylactics.

Alkaloids of Dicranostigma franchetianum (Papaveraceae) and Berberine Derivatives as a New Class of Antimycobacterial Agents.

Tuberculosis (TB) is a widespread infectious disease caused by Mycobacterium tuberculosis. The increasing incidence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains has created a need for new antiTB agents with new chemical scaffolds to combat the disease. Thus, the key question is: how to search for new antiTB and where to look for them? One of the possibilities is to search among natural products (NPs). In order to search for new antiTB drugs, the detailed phytochemical study of the whole Dicranostigma franchetianum plant was performed isolating wide spectrum of isoquinoline alkaloids (IAs). The chemical structures of the isolated alkaloids were determined by a combination of MS, HRMS, 1D, and 2D NMR techniques, and by comparison with literature data. Alkaloids were screened against Mycobacterium tuberculosis H37Ra and four other mycobacterial strains (M. aurum, M. avium, M. kansasii, and M. smegmatis). Alkaloids 3 and 5 showed moderate antimycobacterial activity against all tested strains (MICs 15.625-31.25 µg/mL). Furthermore, ten semisynthetic berberine (16a-16k) derivatives were developed and tested for antimycobacterial activity. In general, the derivatization of berberine was connected with a significant increase in antimycobacterial activity against all tested strains (MICs 0.39-7.81 µg/mL). Two derivatives (16e, 16k) were identified as compounds with micromolar MICs against M. tuberculosis H37Ra (MIC 2.96 and 2.78 µM). All compounds were also evaluated for their in vitro hepatotoxicity on a hepatocellular carcinoma cell line (HepG2), exerting lower cytotoxicity profile than their MIC values, thereby potentially reaching an effective concentration without revealing toxic side effects.

Monoterpene indole alkaloids from Vinca minor L. (Apocynaceae): Identification of new structural scaffold for treatment of Alzheimer's disease.

One undescribed indole alkaloid together with twenty-two known compounds have been isolated from aerial parts of Vinca minor L. (Apocynaceae). The chemical structures of the isolated alkaloids were determined by a combination of MS, HRMS, 1D, and 2D NMR techniques, and by comparison with literature data. The NMR data of several alkaloids have been revised, corrected, and missing data have been supplemented. Alkaloids isolated in sufficient quantity were screened for their in vitro acetylcholinesterase (AChE; E.C. 3.1.1.7) and butyrylcholinesterase (BuChE; E.C. 3.1.1.8) inhibitory activity. Selected compounds were also evaluated for prolyl oligopeptidase (POP; E.C. 3.4.21.26), and glycogen synthase 3ß-kinase (GSK-3ß; E.C. 2.7.11.26) inhibition potential. Significant hBuChE inhibition activity has been shown by (-)-2-ethyl-3[2-(3-ethylpiperidinyl)-ethyl]-1H-indole with an IC50 value of 0.65 ± 0.16 µM. This compound was further studied by enzyme kinetics, along with in silico techniques, to reveal the mode of inhibition. This compound is also predicted to cross the blood-brain barrier (BBB) through passive diffusion.

Amaryllidaceae Alkaloids of Different Structural Types from Narcissus L. cv. Professor Einstein and Their Cytotoxic Activity.

In this detailed phytochemical study of Narcissus cv. Professor Einstein, we isolated 23 previously known Amaryllidaceae alkaloids (1-23) of several structural types and one previously undescribed alkaloid, 7-oxonorpluviine. The chemical structures were identified by various spectroscopic methods (GC-MS, LC-MS, 1D, and 2D NMR spectroscopy) and were compared with literature data. Alkaloids which had not previously been isolated and studied for cytotoxicity before and which were obtained in sufficient amounts were assayed for their cytotoxic activity on a panel of human cancer cell lines of different histotype. Above that, MRC-5 human fibroblasts were used as a control noncancerous cell line to determine the general toxicity of the tested compounds. The cytotoxicity of the tested alkaloids was evaluated using the WST-1 metabolic activity assay. The growth of all studied cancer cell lines was inhibited by pancracine (montanine-type alkaloid), with IC50 values which were in the range of 2.20 to 5.15 µM.

Amaryllidaceae alkaloids from Narcissus pseudonarcissus L. cv. Dutch Master as potential drugs in treatment of Alzheimer's disease.

Twenty-one known Amaryllidaceae alkaloids of various structural types and one undescribed alkaloid, named narcimatuline, have been isolated from fresh bulbs of Narcissus pseudonarcissus L. cv. Dutch Master. The chemical structures were elucidated by combination of MS, HRMS, 1D and 2D NMR spectroscopic techniques, and by comparison with literature data. Narcimatuline amalgamates two basic scaffolds of Amaryllidaceae alkaloids in its core, namely galanthamine and galanthindole. All isolated compounds were evaluated for their in vitro acetylcholinesterase (AChE), butyrylcholinesterase (BuChE), prolyl oligopeptidase (POP), and glycogen synthase kinase-3ß (GSK-3ß) inhibitory activities. The most interesting biological profile was demonstrated by newly isolated alkaloid narcimatuline.

Alkaloids from Narcissus poeticus cv. Pink Parasol of various structural types and their biological activity.

Fifteen Amaryllidaceae alkaloids (1-15) of various structural types were isolated by standard chromatographic methods from fresh bulbs of Narcissus poeticus cv. Pink Parasol. The chemical structures were elucidated by MS, and 1D and 2D NMR spectroscopic analyses, and by comparison with literature data. Narcipavline (5) and narcikachnine (6) are reported here for the first time. In their structure are combined two basic structural types of Amaryllidaceae alkaloids (galanthamine- and galanthindole-structural types), which represent a new structural type of these compounds. Alkaloids isolated in sufficient amounts were evaluated for their human erythrocytic acetylcholinesterase, and human serum butyrylcholinesterase (HuBuChE) inhibition activity using Ellman's method. Z-Gly-Pro-p-nitroanilide was used as substrate in the prolyl oligopeptidase (POP) assay. Untested alkaloids were also screened for their cytotoxic activity against a small panel of human cancer cells, which spanned cell lines from different tissue types. In parallel, MRC-5 human fibroblasts were employed to determine overall toxicity against noncancerous cells. Some compounds were evaluated for their antiprotozoal activity. The newly isolated alkaloid narcipavline (5) showed interesting HuBuChE inhibition activity (IC50 = 24.4 ± 1.2 µM), and norlycoramine (11) demonstrated promising POP inhibition (IC50 = 0.21 ± 0.01 mM).

Anticancer potential of Amaryllidaceae alkaloids evaluated by screening with a panel of human cells, real-time cellular analysis and Ehrlich tumor-bearing mice.

In this study, twenty-two Amaryllidaceae alkaloids were screened for their anticancer potential. All isolates were evaluated for antiproliferative activities on a panel of 17 human cell types of different tissue origin using WST-1 assay. In addition, we determined the antiproliferative effect with a real-time cell analysis xCELLigence system. Thereafter, to evaluate the barely known in vivo anticancer potential of the most potent molecule haemanthamine, a preliminary study was performed using an Ehrlich tumor-bearing mice model. The results showed that haemanthamine, lycorine and haemanthidine exerted the highest antiproliferative activity. The mean growth percent (GP) value after a single-dose 10 µM treatment was for haemanthamine 21%, for lycorine 21% and for haemanthidine 27% that of untreated control cells (100%). Furthermore, haemanthamine, lycorine and haemanthidine exhibited significant cytotoxicities against all the tested cell lines with individual IC50 values in the micromolar range. Dynamic real-time measures of impedance by xCELLigence indicated that these three compounds suppress cell proliferation after 10 h of treatment at a concentration of 10 µM or higher. Regrettably, in a follow-up in vivo antitumor activity study, haemanthamine showed no statistically significant reduction in the tumor size with no prolongation of survival time of Ehrlich tumor-bearing mice. Taken together, these results provide a new clue and guidance for exploiting Amaryllidaceae alkaloids as anticancer agents.

Application of BACE1 immobilized enzyme reactor for the characterization of multifunctional alkaloids from Corydalis cava (Fumariaceae) as Alzheimer's disease targets.

In our ongoing study focused on Corydalis cava (Fumariaceae), used in folk medicine in the treatment of memory dysfunctions, we have investigated fifteen previously isolated alkaloids for their potential multifunctional activity on Alzheimer's disease (AD) targets. Determination of ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibition was carried out using a BACE1-Immobilized Enzyme Reactor (IMER) by validating the assay with a multi-well plate format Fluorescence Resonance Energy Transfer (FRET) assay. Seven alkaloids out of fifteen were found to be active, with (-)-corycavamine (3) and (+)-corynoline (5) demonstrating the highest BACE1 inhibition activity, in the micromolar range, in a concentration dependent manner. BACE1-IMER was found to be a valid device for the fast screening of inhibitors and the determination of their potency. In a permeation assay (PAMPA) for the prediction of blood-brain barrier (BBB) penetration, the most active compounds, (-)-corycavamine (3) and (+)-corynoline (5), were found to be able to cross the BBB. Not all compounds showed activity against glycogen synthase kinase-3ß (GSK-3ß) and casein kinase-1δ (CK-1δ). On the basis of the reported results, we found that some C. cava alkaloids have multifunctional activity against AD targets (prolyl oligopeptidase, cholinesterases and BACE1). Moreover, we tried to elucidate the treatment effectivity (rational use) of its extract in memory dysfunction in folk medicine.

Isoquinoline alkaloids as prolyl oligopeptidase inhibitors.

Prolyl oligopeptidase is a cytosolic serine peptidase that hydrolyses proline-containing peptides at the carboxy terminus of proline residues. It has been associated with schizophrenia, bipolar affective disorder, and related neuropsychiatric disorders and therefore may have important clinical implications. Thirty-one isoquinoline alkaloids of various structural types, previously isolated in our laboratory, were screened for their ability to inhibit prolyl oligopeptidase. Promising results have been showed by alkaloids californidine (IC50=55.6±3.5 µM), dihydrosanquinarine (IC50=99.1±7.6 µM), corypalmine (IC50=128.0±10.5 µM) and N-methyllaurotetanine (IC50=135.0±11.7 µM).

Isoquinoline alkaloids as a novel type of AKR1C3 inhibitors.

AKR1C3 is an important human enzyme that participates in the reduction of steroids and prostaglandins, which leads to proliferative signalling. In addition, this enzyme also participates in the biotransformation of xenobiotics, such as drugs and procarcinogens. AKR1C3 is involved in the development of both hormone-dependent and hormone-independent cancers and was recently demonstrated to confer cell resistance to anthracyclines. Because AKR1C3 is frequently upregulated in various cancers, this enzyme has been suggested as a therapeutic target for the treatment of these pathological conditions. In this study, nineteen isoquinoline alkaloids were examined for their ability to inhibit a recombinant AKR1C3 enzyme. As a result, stylopine was demonstrated to be the most potent inhibitor among the tested compounds and exhibited moderate selectivity towards AKR1C3. In the follow-up cellular studies, stylopine significantly inhibited the AKR1C3-mediated reduction of daunorubicin in intact cells without considerable cytotoxic effects. This inhibitor could therefore be used as a model AKR1C3 inhibitor in research or evaluated as a possible therapeutic anticancer drug. Furthermore, based on our results, stylopine can serve as a model compound for the design and future development of structurally related AKR1C3 inhibitors.

Acetylcholinesterase and butyrylcholinesterase inhibitory compounds from Chelidonium majus (Papaveraceae).

The roots and aerial parts of Chelidonium majus L. were extracted with EtOH and fractionated using CHCl3 and EtOH. Repeated column chromatography, preparative TLC and crystallization led to the isolation of five isoquinoline alkaloids, stylopine (3), chelidonine (4), homochelidonine (5), protopine (6), and allocryptopine (7), along with two isolation artifacts 6-ethoxydihydrosanguinarine (1) and 6-ethoxydihydrochelerythrine (2). All isolated compounds were tested for human blood acetylcholinesterase (HuAChE) and human plasma butyrylcholinesterase (HuBuChE) inhibitory activity. The isolation artifacts exhibited the highest activity against HuAChE and HuBuChE with IC50 values of 0.83 +/- 0.04 microM and 4.20 +/- 0.19 microM for 6-ethoxydihydrochelerythrine and 3.25 +/- 0.24 microM and 4.51 +/- 0.31 microM for 6-ethoxydihydrosanguinarine. The most active of the naturally-occurring alkaloids was chelidonine, which inhibited both HuAChE and HuBuChE in a dose-dependent manner with IC50 values of 26.8 +/- 1.2 microM and 31.9 +/- 1.4 microM, respectively.