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 β-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.

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.

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 3 beta-kinase (GSK-3 beta; E.C. 2.7.11.26) inhibition potential. Significant hBuChE inhibition activity has been shown by (- )-2-ethyl-3[2-(3-ethylpiperidinyl)-ethyl]-1Hindole with an IC50 value of 0.65 +/- 0.16 mu M. 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.

Natural products, especially alkaloids, are still a substantial resource for the drug development [1]. Based on the preliminary screening of alkaloidal extracts of various plants against Alzheimerʼs disease, Vinca minor L. (Apocynaceae) have been chosen for a detailed phytochemical and biological study. V. minor L. is an evergreen trailing subshrub common in Europe with rich content of monoterpene indole alkaloids [2]. In this study, we have isolated and identified active alkaloidal compounds and assessed their potential to inhibit hAChE, hBuChE, POP, and GSK-3β – enzymes that play a key role in the pathophysiology of Alzheimerʼs disease. Using chromatographic methods, we have isolated 23 alkaloids; 11 of them have been reported in this species for the first time. One alkaloidal structure was undescribed and was named as vincaminorudeine. The most active compound was (−)-2-ethyl-3[2-(3-ethylpiperidinyl)-ethyl]-1H-indole with IC50 = 0.65 µM for the inhibition of hBuChE, and with IC50 = 58 µM for the inhibition of POP. Other alkaloids that exhibited significant inhibition against hBuChE (IC50 < 30 µM) were vincaminoreine, minovine, 16-methoxyminovine, vincorine, and tubotaiwine. None of the isolated alkaloids was active against hAChE. The (−)-2-ethyl-3[2-(3-ethylpiperidinyl)-ethyl]-1H-indole was also further studied for its pharmacokinetic, revealing a reversible competitive type of inhibition for hBuChE with Ki = 55 nM. This compound can also penetrate the blood-brain barrier by passive diffusion, as was assessed by the PAMPA study. Additionally, the alkaloid on the panel of ten cell lines showed non-cytotoxicity. These compelling results open a possibility for further research on this indole alkaloid.

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.

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.

The primal discipline from which pharmacy has developed can be considered as pharmacognosy. This review defines pharmacognosy while reflecting on the latest development and discourse about its justifiability in the educational system in pharmaceutical faculties and the history of development of new drugs under the influence of pharmacognosy. The article defines the status quo of the pharmacognosy area, or more precisely its parts (biology, chemistry, production, and technology) and discusses their connections. It underlines the legitimacy of application of natural drugs in therapy, which is undeniable, and proves that whether a new drug was prepared either synthetically or isolated from a natural source is not important. The overview follows the basic requirements of pharmacognosy, especially its methodology (usage of faster and more effective phyto-analytical methods, reverse pharmacology, and reverse pharmacognosy, in silico methods). Pharmacognosy is confronted by three major challenges in the 21st century that can push it significantly forward: ethnopharmacological sources evaluation, evaluation of nutraceuticals, and pharmacognosy of marine organisms. The educational system of universities should correspond to these new demands. However, in some areas the educational system is not prepared to face the challenges of the time. The basic requirement is to adopt a complex attitude to biogenic material and utilize the connections of this complexity in the teaching of modern pharmacy.

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.

Bersavine is the new bisbenzylisoquinoline alkaloid isolated from the Berberis vulgaris L. (Berberidaceae) plant. The results of cytotoxicity screening 48 h post-treatment showed that bersavine 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 bersavine treatment in a time- and dose-dependent manner. Bersavine manifested concentration-dependent antiproliferative activity in human lung, breast, ovarian and hepatocellular carcinoma cell lines using a xCELLigence assay. Significantly higher percentages of MOLT-4 cells exposed to bersavine at 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. The upregulation of p53 phosphorylated on Ser392 was detected during the progression of MOLT-4 cell apoptosis. Mechanistically, bersavine-induced apoptosis is an effect of increased activity of caspases, while reduced proliferation seems dependent on increased Chk1 Ser345 phosphorylation and decreased Rb Ser807/811 phosphorylation in human leukemic cells.

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 a 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-3 beta 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 mu M). The in vitro data were further supported by computational analysis that showed the accommodation of 6 in the active site of hBuChE.

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-3β (GSK-3β) inhibitory activities. The most interesting biological profile was demonstrated by newly isolated alkaloid narcimatuline.

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.

Glycogen synthase kinase-3β (GSK-3β) 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 β-catenin. Recent studies have identified GSK-3β as a potential therapeutic target in Alzheimer´s disease, bipolar disorder, stroke, more than 15 types of cancer, and diabetes. GSK-3β 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-3β. 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)}.

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).

Aldo-keto reductase 103 (AKRIC3) is an important human enzyme that participates in the reduction of steroids and prostaglandins, which leads to proliferative signaling. AKRIC3 is frequently upregulated in various cancers, and this enzyme has been suggested as a therapeutic target for the treatment of these pathological conditions. The fact that the isoquinoline alkaloid stylopine has been identified as a potent AKRIC3 inhibitor has prompted us to screen a library of diverse types of Amaryllidaceae alkaloids, which biogenetically are isoquinoline alkaloids, on a recombinant form of AKRIC3. From the tested compounds, only tazettine showed moderate AKRIC3 inhibitory potency with an IC5o value of 15.8 ? 1.2 pM. Tazettine is a common Amaryllidaceac alkaloid, which could be used as a model substance for the further development of either analogues or related compounds with better inhibition potency.

Plant tissue cultures represent an attractive alternative source of valuable natural substances. Many strategies have been proposed for improving production yields, among others, elicitation with heavy metals. In this work, the effect was investigated of copper(II) sulfate (at concentrations of 0, 0.1, 0.5, 1, 5, 10, 50, 100, and 200 μM) on scopoletin production, as well as on cell growth in Angelica archangelica (angelica) suspension cultures with the cultures maintained in the dark and in the light. Copper(II) sulfate up to 100 μM did not significantly affect either fresh or dry biomass in the cultures grown in either the dark or light, but at 200 μM they were slightly reduced. Copper(II) sulfate stimulated accumulation of scopoletin in a dose-dependent manner both in the cells and in the culture medium. Moreover, concerning scopoletin production, the cultures showed different sensitivity to copper ions depending on light conditions. The highest yields of scopoletin were found at copper concentrations of 5-50 μM and 50 μM in the light- and dark-grown cultures, respectively. Copper(II) sulfate has proved to be a suitable elicitor for improvement of scopoletin accumulation in cell suspension cultures of A. archangelica.

Background: Plants have had crucial role in the folklore of ancient cultures for over 5000 years. In addition to the use as food or spices, plants have also been utilized as medicine. Two remaining living traditions, the traditional Indian and Eastern medicine, have contributed most to the current state of knowledge related to medicinal plants. In their folklore, herbal medicines were prepared e.g. as teas or tinctures. These products are used as complementary treatment alongside conventional drugs till today. Another trend begun in the early 19th cent., which involved the isolation of active compounds from plants. This tendency led to the discovery of the analgesic alkaloids morphine and codeine or the cardiac glycoside digitoxin. Recently, a new alkaloid bersavine was isolated from Berberis vulgaris, along berbamine and berberine. Methods: The dried root bark from Berberis vulgaris was minced and extracted with EtOH. The extract was evaporated, dissolved in HCl and extracted with nonpolar solvent. After the second extraction column chromatography on Al2O3 was performed. Subsequently was executed preparative TLC, which reveal a yet unknown alkaloid, later identified by MS and NMR analysis and named bersavine. Effect of bersavine on viability and proliferation was evaluated by WST assay and Trypan blue staining. Next was analysed its impact on cell cycle and apoptosis using the flow cytometry, activity of caspases and Western blot analysis of regulatory proteins was implemented. Results: Bersavine's effect on cancer cells was first evaluated on panel of 9 different cell lines. Cancer cell lines MOLT-4, Jurkat, HT-29, HeLa and MCF-7 appear to be the most sensitive to the effect of bersavine. Follow-up experiments revealed that bersavine reduced cell viability and proliferation in a dose dependent manner within 24 h of treatment. Moreover, the reduction of cell viability was even more pronounce 48 h following the treatment. The decrease in cell viability was caused by the induction of apoptosis and activation of caspases. Conclusions: All acquired results suggest that bersavine has very promising activity and it would be worthwhile to subject it to further evaluation. Legal entity responsible for the study: Charles University, Faculty of Medicine in Hradec Kralove Funding: Grant Agency of Charles University (Project No. 932616) and SVV-260397/2017. Disclosure: All authors have declared no conflicts of interest.

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.

Two new isoquinoline alkaloids, named fumaranine (2) and fumarostrejdine (10), along with 18 known alkaloids were isolated from aerial parts of Fumaria officinalis. The structures of the isolated compounds were elucidated on the basis of spectroscopic analyses and by comparison with literature data. The absolute configuration of the new compound 2 was determined by comparing its circular dichroism spectra with those of known analogs. Compounds isolated in sufficient amounts were evaluated for their acetylcholinesterase, butyrylcholinesterase, prolyl oligopeptidase (POP), and glycogen synthase kinase‐3β inhibitory activities. Parfumidine (8) and sinactine (15) exhibited potent POP inhibition activities (IC50 99±5 and 53±2 μM, resp.).