Toxicity and mode of action of the aporphine plant alkaloid liriodenine on the insect GABA receptor.

Liriodenine is a biologically active plant alkaloid with multiple effects on mammals, fungi, and bacteria, but has never been evaluated for insecticidal activity. Accordingly, liriodenine was applied topically in ethanolic solutions to adult female Anopheles gambiae, and found to be mildly toxic. Its lethality was synergized in mixtures with dimethyl sulfoxide and piperonyl butoxide. Recordings from the ventral nerve cord of larval Drosophila melanogaster showed that liriodenine was neuroexcitatory and reversed the inhibitory effect of 1 mM GABA at effective concentrations of 20-30 µM. GABA antagonism on the larval nervous system was equally expressed on both susceptible and cyclodiene-resistant rdl preparations. Acutely isolated neurons from Periplaneta americana were studied under patch clamp and inhibition of GABA-induced currents with an IC50 value of about 1 µM were observed. In contrast, bicuculline did not reverse the effects of GABA on cockroach neurons, as expected. In silico molecular models suggested reasonable structural concordance of liriodenine and bicuculline and isosteric hydrogen bond acceptor sites. This study is the first assessing of the toxicology of liriodenine on insects and implicates the GABA receptor as one likely neuronal target, where liriodenine might be considered an active chemical analog of bicuculline.

Discovery of chemical markers for improving the quality and safety control of Sinomenium acutum stem by the simultaneous determination of multiple alkaloids using UHPLC-QQQ-MS/MS.

Sinomenium acutum stem is a popular traditional Chinese medicine used to treat bone and joint diseases. Sinomenine is considered the only chemical marker for the quality control of S. acutum stem in mainstream pharmacopeias. However, higenamine in S. acutum stem is a novel stimulant that was banned by the World Anti-Doping Agency in 2017. Therefore, enhancing the quality and safety control of S. acutum stem to avoid potential safety risks is of utmost importance. In this study, a fast, sensitive, precise, and accurate method for the simultaneous determination of 11 alkaloids in S. acutum stem by ultrahigh-performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry (UHPLC-QQQ-MS/MS) was established. This method successfully analyzed thirty-five batches of S. acutum stem samples. The average contents of sinomenine, magnoflorine, coclaurine, acutumine, higenamine, sinoacutine, palmatine, magnocurarine, columbamine, 8-oxypalmatine, and jatrorrhizine were 24.9 mg/g, 6.35 mg/g, 435 µg/g, 435 µg/g, 288 µg/g, 44.4 µg/g, 22.5 µg/g, 21.1 µg/g, 15.8 µg/g, 9.30 µg/g, and 8.75 µg/g, respectively. Multivariate analysis, including principal component analysis (PCA), orthogonal partial least square method-discriminant analysis (OPLS-DA), and hierarchical cluster analysis (HCA), were performed to characterize the importance and differences among these alkaloids in S. acutum stem samples. As a result, sinomenine, magnoflorine, coclaurine, acutumine, and higenamine are proposed as chemical markers for quality control. Higenamine and coclaurine are also recommended as chemical markers for safety control. This report provides five alkaloids that can be used as chemical markers for improving the quality and safety control of S. acutum stem. It also alerts athletes to avoid the risks associated with consuming S. acutum stem.

Magnoflorine: A review of its pharmacology, pharmacokinetics and toxicity.

Magnoflorine is an important quaternary aporphine alkaloid that is isolated from some commonly used herbal medicines (e.g., Sinomenium acutum (Thunb.) Rehder & E.H.Wilson and Coptis chinensis Franch.). In recent years, magnoflorine has received increasing attention due to its multiple pharmacological activities. This review provides the first comprehensive summary of the plant sources, pharmacological effects, toxicity, and pharmacokinetic characteristics of magnoflorine. The results indicated that magnoflorine possesses a wide spectrum of pharmacological properties, including anti-diabetic, anti-inflammatory, neuropsychopharmacological, immunomodulatory, hypotensive, antioxidant, and antifungal activities. Pharmacokinetic studies have shown that magnoflorine has low bioavailability and high absorption and elimination rates. However, the other compounds (e.g., berberine) present in herbal medicines could reduce the absorption and removal rates of magnoflorine and increase its bioavailability. Moreover, toxicity studies have suggested that magnoflorine is non-toxic to most cells. However, long-term and high-dose toxicity testing in animals is still lacking. In view of good pharmacological activities, magnoflorine is expected to be a potential drug candidate for the treatment of diabetes, depression, or Alzheimer's disease. However, further studies are needed to elucidate its molecular mechanisms and targets, clarify its toxicity, and improve its oral bioavailability.

Boldine Ameliorates Vascular Oxidative Stress and Endothelial Dysfunction: Therapeutic Implication for Hypertension and Diabetes.

Epidemiological and clinical studies have demonstrated that a growing list of natural products, as components of the daily diet or phytomedical preparations, are a rich source of antioxidants. Boldine [(S)-2,9-dihydroxy-1,10-dimethoxy-aporphine], an aporphine alkaloid, is a potent antioxidant found in the leaves and bark of the Chilean boldo tree. Boldine has been extensively reported as a potent "natural" antioxidant and possesses several health-promoting properties like anti-inflammatory, antitumor promoting, antidiabetic, and cytoprotective. Boldine exhibited significant endothelial protective effect in animal models of hypertension and diabetes mellitus. In isolated thoracic aorta of spontaneously hypertensive rats, streptozotocin-induced diabetic rats, and db/db mice, repeated treatment of boldine significantly improved the attenuated acetylcholine-induced endothelium-dependent relaxations. The endothelial protective role of boldine correlated with increased nitric oxide levels and reduction of vascular reactive oxygen species via inhibition of the nicotinamide adenine dinucleotide phosphate oxidase subunits, p47 and nicotinamide adenine dinucleotide phosphate oxidase 2, and angiotensin II-induced bone morphogenetic protein-4 oxidative stress cascade with downregulation of angiotensin II type 1 receptor and bone morphogenetic protein-4 expression. Taken together, it seems that boldine may exert protective effects on the endothelium via several mechanisms, including protecting nitric oxide from degradation by reactive oxygen species as in oxidative stress-related diseases. The present review supports a complimentary therapeutic role of the phytochemical, boldine, against endothelial dysfunctions associated with hypertension and diabetes mellitus by interfering with the oxidative stress-mediated signaling pathway.

Anti-invasion effect of crebanine and O-methylbulbocapnine from Stephania venosa via down-regulated matrix metalloproteinases and urokinase plasminogen activator.

The alkaloids isolated from Stephania venosa (S. venosa) have been shown to inhibit the proliferation and to induce the apoptosis of cancer cells. However, the anti-metastatic effect of the alkaloids on cancer cell invasion is unknown. In this study, we investigated the anti-invasive properties of four alkaloids from S. venosa, crebanine (CN), O-methylbulbocapnine (OMBC), tetrahydropalmatine (THP), and N-methyltetrahydropalmatine (NMTHP), in HT1080 human fibrosacroma cells. Treatment of the cells with 15 µg/mL of CN and OMBC reduced the chemo-invasion of HT1080 cells to 45 and 50%, respectively, whereas THP and NMTHP had a negative effect. On the other hand, CN and OMBC had no effect on cell migration. Matrix metalloproteinases (MMPs) and urokinase plasminogen activator (uPA) are the extracellular matrix (ECM) degradation enzymes that play an important role in cancer cell metastasis. Results from zymography and western blot analysis showed that CN and OMBC comparatively reduced MMP-2, MMP-9, MT1-MMP and uPA expression in a dose-dependent manner. However, CN and OMBC had no effect on the activity of collagenase, MMP-2 and MMP-9. We also found that CN and OMBC reduced the nuclear translocation and DNA binding activity of nuclear factor kappa B (NF-κB), which is the expressed mediator of ECM degradation enzymes. These findings demonstrated that CN and OMBC mediated HT1080 cell invasion by the reduction of MMP-2, MMP-9, uPA and MT1-MMP expression, possibly by targeting of NF-κB signaling pathway in the HT1080 cells.

Studies on the metabolism and toxicological detection of glaucine, an isoquinoline alkaloid from Glaucium flavum (Papaveraceae), in rat urine using GC-MS, LC-MS(n) and LC-high-resolution MS(n).

Glaucine ((S)-5,6,6a,7-tetrahydro-1,2,9,10-tetramethoxy-6-methyl-4H-dibenzo [de,g]quinoline) is an isoquinoline alkaloid and main component of Glaucium flavum (Papaveraceae). It was described to be consumed as recreational drug alone or in combination with other drugs. Besides this, glaucine is used as therapeutic drug in Bulgaria and other countries as cough suppressant. Currently, there are no data available concerning metabolism and toxicological analysis of glaucine. To study both, glaucine was orally administered to Wistar rats and urine was collected. For metabolism studies, work-up of urine samples consisted of protein precipitation or enzymatic cleavage followed by solid-phase extraction. Samples were afterwards measured by liquid chromatography (LC) coupled to low or high-resolution mass spectrometry (HR-MS). The phase I and II metabolites were identified by detailed interpretation of the corresponding fragmentations, which were further confirmed by determination of their elemental composition using HR-MS. From these data, the following metabolic pathways could be proposed: O-demethylation at position 2, 9 and 10, N-demethylation, hydroxylation, N-oxidation and combinations of them as well as glucuronidation and/or sulfation of the phenolic metabolites. For monitoring a glaucine intake in case of abuse or poisoning, the O- and N-demethylated metabolites were the main targets for the gas chromatography-MS and LC-MS(n) screening approaches described by the authors. Both allowed confirming an intake of glaucine in rat urine after a dose of 2 mg/kg body mass corresponding to a common abuser's dose.

Applied biological and physicochemical activity of isoquinoline alkaloids: oxoisoaporphine and boldine.

The aim of this study was to determine the electronic influence of substituent groups and annelated rings such as oxazole-oxazinone on the physicochemical and photoprotection, antioxidant capacity, toxicity and singlet oxygen photosensitization biological properties of isoquinoline alkaloid frameworks. Thus, oxoisoaporphine derivatives 1-5 and 3-azaoxoisoaporphine (6), some of them with phenolic structures, did not present any antioxidant capacity, possibly either by formation of keto-enol tautomerism species or the formation of unstable free radicals. Due to the singlet oxygen quantum yields (FD) near to unity, and greater photostability than phenalenone, oxoisoaporphines 4-6 may be considered as photosensitizers for singlet oxygen production and can be used as new universal study tools. The biological application as antibacterial agents is an important and possible tool in the study of compounds with low cytotoxicity and high reactivity in antineoplastic chemotherapy. On the other hand, when boldine and its annelated derivatives B1-4 are irradiated, a photoprotector effect is observed (SPF = 2.35), even after 30 minutes of irradiation. They also act as photoprotectors in cell fibroblast cultures. No hemolysis was detected for boldine hydrochloride and its salts without irradiation. In solutions irradiated before incubation (at concentrations over 200 ppm) photoproducts were toxic to the nauplii of Artemia salina.

Cytotoxicity of aporphines in human colon cancer cell lines HCT-116 and Caco-2: an SAR study.

A series of synthetic aporphine derivatives structurally related to domesticine and nantenine (ring A, N6 and ring C truncated analogs), was evaluated in MTS cytotoxicity assays against the human colon cancer cell lines, HCT-116 and Caco-2. In general, the C1 position of ring A is tolerant of alkoxy substituents as well as a benzoyl ester functionality. Other modifications evaluated resulted in a decrease in cytotoxic activity. The most potent compounds identified had IC(50) values in the range 23-38 µM, comparable to the known cytotoxic agent, etoposide.

Chemical investigation of drug-like compounds from the Australian tree, Neolitseadealbata.

Two of the four parameters in the 'rule of five', molecular weight and logP, which can be detected and predicted by mass spectrometry and compound retention on reversed-phase HPLC, were used as guidelines in natural product isolation. A new aporphine alkaloid, (6aR)-normecambroline (1), was isolated from the bark of Neolitsea dealbata (R. Br.) Merr. Its structure was determined on the basis of NMR, MS and CD analysis. It is the first time the absolute configuration of the roemerine-N-oxide was assigned for both roemerine-N(α)-oxide (3) and roemerine-N(ß)-oxide (4). Physico-chemical property evaluation demonstrated all alkaloids had no Lipinski violation. Compound 1 inhibited selectively against cervical cancer cells (HeLa) with an IC(50) of 4.0 µM.

Divalent later transition metal complexes of the traditional chinese medicine (TCM) liriodenine: coordination chemistry, cytotoxicity and DNA binding studies.

Liriodenine (L), a natural alkaloid, was isolated as an active component from the anticancer traditional Chinese medicine (TCM), Zanthoxylum nitidum. It reacted with Mn(II), Fe(II), Co(II) and Zn(II) to afford four metal complexes: [MnCl(2)(L)(2)] (1), [FeCl(2)(L)(2)] (2), [Co(L)(2)(H(2)O)(2).Co(L)(2)(CH(3)CH(2)OH)(2)](ClO(4))(4) (3), and [Zn(2)(L)(2)(mu(2)-Cl)(2)Cl(2)] (4), which were characterized by elemental analysis, IR, ESI-MS. Their crystal structures were determined by the single crystal X-ray diffraction method. The in vitro cytotoxicity of L and complexes 1-4 against 10 human tumour cell lines was assayed. Some of these metal-based compounds exhibited enhanced cytotoxicity vs. free L to selected tumour cell lines. The binding properties of L and its complexes 1-4 to ct-DNA were investigated by spectroscopic methods and viscosity measurements. Agarose gel electrophoresis experiments were also carried out to evaluate their unwinding ability towards plasmid DNA and their inhibition towards Topoisomerase I. All the results indicate that complexes 1-4 may bind more intensively to the DNA helix than does L, and intercalative binding for complexes 1-4 and electrostatic interactions for complexes 3-4 to DNA should be considered. For complex 4, covalent binding to DNA may exist. Of special note, all these metal complexes effectively inhibit Topoisomerase I even at low concentration (< or = 10 microM).

Secondary metabolites and cytotoxic activities from the stem bark of Zanthoxylum nitidum.

A dihydrobenzo[c]phenanthridine alkaloid, epizanthocadinanine A (1), together with 27 known compounds, including eight benzo[c]phenanthridines, i.e., oxynitidine (2), oxyavicine (3), oxychelerythrine (4), dihydrochelerythrine (5), 6-acetonyldihydrochelerythrine (6), norchelerythrine (7), decarine (8), and arnottianamide (9); two 2-quinolones, i.e., flindersine (10) and 4-methoxy-1-methyl-2-quinolone (11); two furoquinolines, i.e., skimmianine (12) and gamma-fagarine (13); three aporphines, i.e., liriodenine (14), N-acetyldehydroanonaine (15), and N-acetylanonaine (16); six lignans, i.e., sesamin (17), episesamin (18), piperitol-3,3-dimethylallyl ether (19), xanthoxylol-3,3-dimethylallyl ether (20), savinin (21), and 2,3-bis(3,4-methylenedioxybenzyl)but-2-en-4-olide (22); three terpenoids, i.e., alpha-cadinol (23), anticopalol (24), and spathulenol (25); one coumarin, i.e., aesculetin dimethyl ether (26); and two steroids, i.e., beta-sitosterol (27) and beta-sitostenone (28) were isolated from the stem bark of Zanthoxylum nitidum. Their structures were elucidated on the basis of extensive 1D- and 2D-NMR as well as MS analyses. Moreover, the recently reported structures 2'-4' of rhoifolines B and A, and '8-methoxynorchelerythrine', resp., isolated as new compounds from Z. rhoifolium and Z. nitidum, resp., could be assigned the revised structures 2-4 by reinvestigation of the spectroscopic data. In addition, the cytotoxicity of the isolates was evaluated on the MCF-7, NCI-H460, and SF-268 cell lines. Among these isolates, liriodenine (14) was the most active compound against the MCF-7, NCI-H460, and SF-268 cell lines with IC(50) values of 2.19, 2.38, and 3.19 microg/ml, resp.

Synthesis of aristolactam analogues and evaluation of their antitumor activity.

A series of natural aristolactams and their analogues have been prepared and evaluated for antitumor activity against human cancer cells, including multi-drug resistant cell lines. Naturally occurring aristolactams, such as aristolactam BII (cepharanone B), aristolactam BIII, aristolactam FI (piperolactam A), N-methyl piperolactam A, and sauristolactam showed moderate antitumor activities in selected cell lines. However, several synthetic aristolactam derivatives exhibited potent antitumor activities against a broad array of cancer cell lines with GI(50) values in the submicromolar range.

Toxicological evaluation of the hydro-alcohol extract of the dry leaves of Peumus boldus and boldine in rats.

The hydro-alcohol extract of the dry leaves of Peumus boldus and boldine, showed abortive and teratogenic action and changes in the blood levels of bilirubin, cholesterol, glucose, alanine aminotransferase (ALT), aspartate aminotransferase (AST) and urea in rats. The long term administration of the extract and boldine did not cause histological modification during a period of 90 days.

Phenolic aporphine-benzylisoquinoline alkaloids from Thalictrum faberi.

From the roots of Thalictrum faberi, six new phenolic aporphine-benzylisoquinoline alkaloids, 3-hydroxy-6'-desmethyl-9-O-methylthalifaboramine (1), 3-hydroxythalifaboramine (2), 6'-desmethylthalifaboramine (3); 3,5'-dihydroxythalifaboramine (4), 5'-hydroxythalifaboramine (5) and 3-hydroxy-6'-desmethylthalifaboramine (6) were isolated. Their structures were established through the use of one- and two-dimensional NMR techniques. All of the tested alkaloids showed potent cytotoxic and antimalarial activities.

Evaluation of the genotoxic potential of the alkaloid boldine in mammalian cell systems in vitro and in vivo.

Boldine is an alkaloid present in Peumus boldus (popularly called "boldo-do-chile" in Brazil) which has healing properties and is used for the treatment of gastrointestinal disorders. The possible clastogenic effect of the drug was tested in vitro on human peripheral blood lymphocytes by evaluating the induction of chromosome aberrations and sister-chromatid exchanges (SCEs). Cultures from different individuals were treated with boldine at concentrations of 10, 20 and 40 micrograms/ml of culture medium. The effect of the alkaloid was also tested in an in vivo assay using BALB/c mouse bone marrow cells. Boldine was administered to the animals by gavage at the concentrations of 225, 450 and 900 mg/kg body weight. Under the conditions used, boldine did not induce a statistically significant increase in the frequency of chromosome aberrations or SCEs in either test system.

Central action of glaucine in rodents.

It was found that glaucine (7, 14 and 28 mg/kg ip) decreased locomotor activity, did not affect the body temperature and potentiated the amphetamine and apomorphine stereotypy but did not affect the amphetamine hypermotility. Besides, glaucine antagonized haloperidol-induced catalepsy of the rats. Glaucine was without analgesic, anticonvulsant, antianxiety and antidepressive effects in mice. This alkaloid in doses of 14 and 28mg/kg ip antagonized the head twitch response induced by 5-HTP in the rats. Further, glaucine decreased the hyperthermia induced by m-CPP in rats kept at high ambient temperature. It was found that glaucine depressed the noradrenaline (NA) and dopamine (DA) levels in the whole brain of mice. Besides, this alkaloid significantly decreased the brain level of 5-hydroxytryptamine (5-HT), without changing the 5-hydroxyindoleacetic acid (5-HIAA) concentration.

Genotoxicity of the boldine aporphine alkaloid in prokaryotic and eukaryotic organisms.

The aporphine alkaloid boldine, present in Peumus boldus (boldo-do-Chile) widely used all over the world, was tested for the presence of genotoxic, mutagenic and recombinogenic activities in microorganisms. This alkaloid did not show genotoxic activity with or without metabolic activation in the SOS chromotest and Ames tester strains TA100, TA98 and TA102. It was not able to induce point and frameshift mutations in haploid Saccharomyces cerevisiae cells. However, mitotic recombinational events such as crossing-over and gene conversion were weakly induced in diploid yeast cells by this alkaloid. Also, boldine was able to induce weakly cytoplasmic 'petite' mutation in haploid yeast cells.

Mutagenicity of isoquinoline alkaloids, especially of the aporphine type.

The mutagenicity of 44 isoquinoline alkaloids was tested in Salmonella typhimurium TA100 and TA98 in the presence or absence of S9 mix. The alkaloids tested included compounds from the isoquinoline, benzylisoquinoline, bisbenzylisoquinoline, monoterpene isoquinoline, berberine, morphinane, hasubanan, benzo[c]phenanthridine and aporphine groups. Among the alkaloids tested, liriodenine was the most potent mutagen for TA100 and roemerine was the most potent for TA98. A clear structure-mutagenicity relationship was observed in a series of aporphine alkaloids (aporphine, dehydroaporphine, 7-oxoaporphine and 4,5-dioxoaporphine), and 10,11-non-substituted aporphines were suggested to exert their mutagenicity through metabolic activation of the 10,11 positions, possibly as the 10,11-epoxides.

[Cytogenetic effect of thaliblastine in a culture of human peripheral blood lymphocytes]. / Tsitogenetichekii éffekt taliblastina v kul'ture limfotsitov perifericheskoi krovi cheloveka.

The mutagenicity of thaliblastine (Bulgarian potential antitumor drug) was investigated in vitro in lymphocytes from healthy donors, and in vivo in lymphocytes of oncological patients after thaliblastine administration. No increase in the rate of chromosome aberrations was noted with increasing thaliblastine concentrations in vitro and in the course of therapy in vivo. Some polyploid metaphases were found in the lymphocytes of the patients treated with thaliblastine, as a result of the statmokinetic effect of the drug. Thaliblastine exerts extraordinarily slight mutagenic effect, as compared with other cytostatics.