Effect of alkaloids from celandine on calcium accumulation and oxidative phosphorylation in mitochondria depending on their DNA intercalating properties

The effect of principal alkaloids (sanguinarine, chelerythrine, coptisine, chelidonine) of greater celandine Chelidonium majus L., as well as the alkaloids from Colchicum autumnale L. (colchicine and colchamine) on calcium accumulation and oxidative phosphorylation in rat liver mitochondria has been studied. The obtained data were compared with DNA intercalating properties of alkaloids detected by the method of thermodenaturation (DNA melting curve plots). It was found that chelerythrine and sanguinarine blocked absorption and accumulation of calcium cations and inhibited oxidative phosphorylation, while the coptisine significantly diminished those indices. Chelidonine, colchicines and colchamine had no influence on the studied characteristics. The effect of alkaloids upon mitochondria functional state correlated tightly with their DNA intercalating properties: chelerythrine and sanguinarine were strong intercalators, while coptisine was a weak one, and chelidonine, colchicine and colchamine did not interact with DNA and caused no changes in its melting point. Correlation coefficient between the intercalating properties of alkaloids and their inhibition of calcium accumulation was 0.89, and with their oxidative phosphorylation inhibition - 0.93. It is suggested that the effect of studied alkaloids upon functional properties of mitochondria can be mediated by mtDNA.     

The inhibition enzymatic hydrolysis of acetylthiocholine by acetylcholinesterase using principal alkaloids isolated from celandine and macleya and their derivatives

A study was made of a possible inhibitory action on the enzymatic hydrolysis of acetylthiocholine by human erythrocyte acetylcholinesterase of principal alkaloids isolated from Chelidonium majus L. and Macleaya (Bocconia) cordata and microcarpa (namely sanguinarine, chelidonine, berberine), and of drugs "Ukrain" (thiophosphoric acid derivative of a sum of the alkaloids isolated from Chelidonium majus L.) and "Sanguirythrine" (a mixture of unseparated closely related to benzo[c]phenanthridine alkaloids sanguinarine and chelerythrine, isolated from Chelidonium majus L. and other plants of Papaveraceae family). All agents under study have been shown to be reversible inhibitors of the enzymatic hydrolysis of acetylthiocholine. On the basis of the kinetic data it has been determined that chelidonine belonged to reversible inhibitors of a competitive type. All other examined agents have been demonstrated to be inhibitors of a mixed competitive-noncompetitive type, and a greater contribution to the inhibition was made by the competitive constituent. Among all examined agents berberine, sanguinarine and "Sanguirythrine" were the strongest inhibitors of this reaction (the values of generalized inhibitory constants being 0.23, 0.23 and 0.29 microM, respectively) and cheliodonine and "Ukrain" were much weaker (2.0 and 2.5 microM, respectively). Judging from the data obtained, sanguinarine and chelerythrine exert similar inhibitory effects on the reaction of enzymatic hydrolysis of acetylthiocholine, since sanguinarine and "Sanguirythrine" have nearly equal generalized inhibitory constants.     

Inhibition of liver mitochondrial monoamine oxidase activity by alkaloids isolated from Chelidonium and Macleaya and by their derivative drugs

It has been shown that the major alkaloids from plants Chelidonium majus L. and Macleaya (Bocconia) cordata and microcarpa, namely, berberine, sanguinarine, chelidonine, and drugs "Ukrain" (thiophosphoric acid derivative of a sum of the alkaloids isolated from Ch. majus L.) and "Sanguirythrine" (a mixture of the alkaloids sanguinarine and chelerythrine, w/w 3:7, isolated from Macleaya), are irreversible inhibitors of oxidative deamination reaction of serotonin and tyramine as substrates, catalyzed by rat liver mitochondrial monoamine oxidase (MAO). At the same time these substances do not influence the oxidative deamination reaction of benzylamine as substrate (in concentration 1 mM or less). The substrate specificity of this inhibition manifests that mainly the oxidative deamination reactions catalyzed by MAO form A are inhibited by the agents studied. Among the examined agents, alkaloid chelidonine and drug "Ukrain" are the strongest inhibitors of the reaction. Alkaloids berberine and sanguinarine and drug "Sanguirythrine" exhibit a weaker action. Judging from the data obtained, sanguinarine and chelerythrine appear to exert similar inhibitory effects in this reaction, since sanguinarine and "Sanguirythrine" have similar values of bimolecular rate constants of their interaction with mitochondrial MAO. As it is well known, the MAO inhibitors appear to be, as a rule, pronounced antidepressants. The combination of malignotoxicity and antidepressive activity in drug "Ukrain" seems to be favourable for its clinical applications.     

Differential effect of sanguinarine, chelerythrine and chelidonine on DNA damage and cell viability in primary mouse spleen cells and mouse leukemic cells

Sanguinarine, chelerythrine and chelidonine are isoquinoline alkaloids derived from the greater celandine. They possess a broad spectrum of pharmacological activities. It has been shown that their anti-tumor activity is mediated via different mechanisms, which can be promising targets for anti-cancer therapy. We focused our study on the differential effects of these alkaloids upon cell viability, DNA damage effect and nucleus integrity in mouse primary spleen cells and mouse lymphocytic leukemic cells, L1210. Sanguinarine and chelerythrine produce a dose-dependent increase in DNA damage and cytotoxicity in both primary mouse spleen cells and L1210 cells. Chelidonine did not show a significant cytotoxicity or damage DNA in both cell types, but completely arrested growth of L1210 cells. Examination of nuclear morphology revealed more cells with apoptotic features upon treatment with chelerythrine and sanguinarine, but not chelidonine. In contrast to primary mouse spleen cells, L1210 cells showed slightly higher sensitivity to sanguinarine and chelerythrine treatment. This suggests that cytotoxic and DNA damaging effects of chelerythrine and sanguinarine are more selective against mouse leukemic cells and primary mouse spleen cells, whereas chelidonine blocks proliferation of L1210 cells. The action of chelidonine on normal and tumor cells requires further investigation.     

Chemotaxonomy and Resource Utilization of the Tribe Chelidonieae (Papaveraceae)

The tribe Chelidonieae of Papaveraceae, consists of 8 genera and 23 species in the world. Most species belonging to this taxon contain dominantly biologically active substances and are widely used as medicinal plants. A chemotaxonomic study in this tribe reveals that Chelidonieae is linked to the other tribes in the presence of protopine-type alkaloids, and the ubiquitous occurrence of dehydrogenated benzophenanthridine-type alkaloids (ex. chelerythrine and sanguinarine) can be served as a chemical character of Chelidonieae. Furthermore, our results indicate that Chelidonieae can obviously be divided into two groups:the first group, including genera Sanguinaria, Eomecon, Macleaya and Bocconia, is characterized by the absence of TLC detected aporphine-, tetrahydroberberine- and reduced benzophenanthridi netype alkaloids, and their undulatedly or palmately incised leaves, as well as short capsules. The second group, comprising Stylophorum, Hylomecon, Dicranostigma and Chelidonium, yields predominately tetrahydroberberine- and reduced benzophenanthridinetype alkaloids, with usually deeply pinnatifid leaves, and long and slender capsules. As regards the medical importance and the resource utilization of Chelidonieae much attention should be paid to the following alkaloids and the taxa: chelidonine (Chelidonium majus ) , isocorydine ( Dicranostigma ) , tetrahydrocoptisine ( Stylophorum ) , chelerythrine (whole tribe particularly the genus Macleaya) and sanguinarine (whole tribe, particularly Macleaya).     

Effect of some isoquinoline alkaloids on enzymatic activity of acetylcholinesterase and monoamine oxidase

It has been shown, that some benzo[c]-phenanthridine and diisoquinoline alkaloids isolated from Chelidonium majus L. and Macleaya (Bocconia) cordata and M. microcarpa (berberine, sanguinarine, chelidonine) and of drugs ("Ukrain" and "Sanguirythrine") inhibited the enzyme activity of acetylcholinesterase from human erythrocyte and monoamine oxidase from the rat liver. All agents under study have been shown to be reversible inhibitors of the enzymatic hydrolysis of acetylthiocholine. It has been determined that chelidonine belonged to reversible inhibitors of a competitive type, all other examined agents have been demonstrated to be inhibitors of a mixed competitive-noncompetitive type, and a greater contribution to the inhibition was made by the competitive constituent. Among all examined agents berberine, sanguinarine and "Sanguirythrine" were the strongest inhibitors of this reaction and chelidonine and "Ukrain" were much weaker. All agents under study have been shown to be irreversible inhibitors of the oxidative deamination reaction of serotonine and tyramine and not to influence the oxidative deamination reaction of benzylamine as a substrate. Among the examined agents, alkaloid sanguinarine and drug "Ukrain" are the strongest inhibitors of the reaction, alkaloids berberine, sanguinarine and "Sanguirythrine" exhibit a weaker action.     

Differential effect of the benzophenanthridine alkaloids sanguinarine and chelerythrine on glycine transporters

Glycine transporter inhibitors modulate the transmission of pain signals. Since it is well known that extracts from medicinal plants such as Chelidonium majus exhibit analgesic properties, we investigated the effects of alkaloids typically present in this plant on glycine transporters. We found that chelerythrine and sanguinarine selectively inhibit the glycine transporter GlyT1 with comparable potency in the low micromolar range while berberine shows no inhibition at all. At this concentration both alkaloids only minimally affected transport of the closely related glycine transporter GlyT2, suggesting that the effect is not mediated by the inhibitory activity of these alkaloids on the Na(+)/K(+) ATPase. GlyT1 inhibition was time-dependent, noncompetitive and increased with glycine concentration. While chelerythrine inhibition was reversible, the effect of sanguinarine was resistant to wash out. These results suggest that benzophenanthridine alkaloids interact with glycine transporters and at low micromolar range selectively target glycine transporter GlyT1.     

Interaction of benzo[c]phenanthridine and protoberberine alkaloids with animal and yeast cells

We compared the effects of four quaternary benzo[c]phenanthridine alkaloids – chelerythrine, chelilutine, sanguinarine, and sanguilutine – and two quaternary protoberberine alkaloids – berberine and coptisine – on the human cell line HeLa (cervix carcinoma cells) and the yeastsSaccharomyces cerevisiae andSchizosaccharomyces japonicus var. versatilis. The ability of alkaloids to display primary fluorescence, allowed us to record their dynamics and localization in cells. Cytotoxic, anti-microtubular, and anti-actin effects in living cells were studied. In the yeasts, neither microtubules nor cell growth was seriously affected even at the alkaloid concentration of 100 μg/ml. The HeLa cells, however, responded to the toxic effect of alkaloids at concentrations ranging from 1 to 50 μg/ml. IC₅₀ values for individual alkaloids were: sanguinarine IC₅₀ = 0.8 μg/ml, sanguilutine IC₅₀ = 8.3 μg/ml, chelerythrine IC₅₀ = 6.2 μg/ml, chelilutine IC₅₀ = 5.2 μg/ml, coptisine IC₅₀ = 2.6 μg/ml and berberine IC₅₀ >10.0 μg/ml. In living cells, sanguinarine produced a decrease in microtubule numbers, particularly at the cell periphery, at a concentration of 0.1 μg/ml. The other alkaloids showed a similar effect but at higher concentrations (5–50 μg/ml). The strongest effects of sanguinarine were explained as a consequence of its easy penetration through the cell membrane owing to nonpolar pseudobase formation and to a high degree of molecular planarity. This revised version was published online in July 2006 with corrections to the Cover Date.     

DNA adduct formation from quaternary benzo[c]phenanthridine alkaloids sanguinarine and chelerythrine as revealed by the P-postlabeling technique

Using the ³²P-postlabeling assay, we investigated the ability of quaternary benzo[c]phenanthridine alkaloids, sanguinarine, chelerythrine and fagaronine, to form DNA adducts in vitro. Two enhanced versions of the assay (enrichment by nuclease P1 and 1-butanol extraction) were utilized in the study. Hepatic microsomes of rats pre-treated with β-naphthoflavone or those of uninduced rats, used as metabolic activators, were incubated in the presence of calf thymus DNA and the alkaloids, with NADPH used as a cofactor. Under these conditions sanguinarine and chelerythrine, but not fagaronine, formed DNA adducts detectable by ³²P-postlabeling. DNA adduct formation by both alkaloids was found to be concentration dependent. When analyzing different atomic and bond indices of the C₁₁---C₁₂ bond (ring B) in alkaloid molecules we found that fagaronine behaved differently from sanguinarine and chelerythrine. While sanguinarine and chelerythrine showed a preference for electrophilic attack indicating higher potential to be activated by cytochrome P450, fagaronine exhibited a tendency for nucleophilic attack. Our results demonstrate that sanguinarine and chelerythrine are metabolized by hepatic microsomes to species, which generate DNA adducts.     

Sanguinarine reductase, a key enzyme of benzophenanthridine detoxification

Cultured cells of Eschscholzia californica respond to a yeast glycoprotein elicitor by producing benzophenanthridine alkaloids, which are excreted into the cell wall and the outer medium. These compounds, preferentially sanguinarine, are efficient phytoalexins because of their ability to intercalate double-stranded DNA (dsDNA), penetrate membranes and inhibit various enzymes containing SH-groups. Externally added sanguinarine is rapidly taken up by intact cells and converted to dihydrosanguinarine, which is substituted intracellularly according to the biosynthetic route. A 29.5 kDa soluble enzyme that catalyses the reduction of sanguinarine and chelerythrine by either NADPH or NADH has been isolated and purified to homogeneity. Benzophenanthridines that accumulate in the outer medium, mainly 10-OH-chelerythrine, chelirubine and macarpine, are converted by the isolated enzyme and by intact cells at much slower rates than sanguinarine. The cellular capacity of uptake and conversion of sanguinarine largely surpasses the rate of alkaloid production. We conclude that the sanguinarine produced by intact cells, after excretion and binding to cell wall elements, is rapidly reabsorbed and reduced to the less toxic dihydrosanguinarine, which then undergoes further biosynthetic reactions. This recycling process would allow the presence of the toxic phytoalexin at the cellular surface without taking the risk of injuring the producing cell.     

Induction of respiration-deficient mutants in Saccharomyces cerevisiae by chelerythrine

Abstract Chelerythrine and sanguinarine, two structurally related benzo/c/phenanthridine alkaloids, prevented growth of yeast cells in medium containing either glucose or non-fermentable carbon sources. At concentrations permitting growth of the yeast Saccharomyces cerevisiae , chelerythrine, but not sanquinarine, induced cytoplasmic respiration-deficient mutants. The petite clones that were analysed exhibited suppressiveness and contained different fragments of the wild-type mitochondrial genome.     

Mitodepressive effects ofChelidonium alkaloids on root tip cells ofAllium cepa L

A comparison of the effects of a water extract of the fresh vegetation apex ofChelidonium majus L. var.majus, with those of berberine sulphate and of chelidonine hydrochloride is reported. The water extract of fresh stems and leaves ofChelidonium majus and the berberine sulphate solution had marked mitodepressive effects on onion root tip cells. Chelidonine hydrochloride had similar but less marked effects. On the basis of the results obtained it can be assumed that the most active group of substances with cytostatic effects, hindering the cells from entering mitosis, are protoberberine bases contained in the latex ofChelidonium majus. Within the range of the investigated concentrations the extract of fresh stems and leaves was less toxic for the cells than berberine sulphate. The data ascertained provide evidence that the mechanism of the cytostatic action of chelidonine, of berberine, as well as of the extract of fresh stems and leaves ofChelidonium, majus differs from the mechanism of the action of colchicine. Of the testedChelidonium alkaloids only chelidonine produced a partial inactivation of the mitotic spindle.     

DNA adduct formation from quaternary benzo[c]phenanthridine alkaloids sanguinarine and chelerythrine as revealed by the 32P-postlabeling technique

Using the 32P-postlabeling assay, we investigated the ability of quaternary benzo[c]phenanthridine alkaloids, sanguinarine, chelerythrine and fagaronine, to form DNA adducts in vitro. Two enhanced versions of the assay (enrichment by nuclease P1 and 1-butanol extraction) were utilized in the study. Hepatic microsomes of rats pre-treated with beta-naphthoflavone or those of uninduced rats, used as metabolic activators, were incubated in the presence of calf thymus DNA and the alkaloids, with NADPH used as a cofactor. Under these conditions sanguinarine and chelerythrine, but not fagaronine, formed DNA adducts detectable by 32P-postlabeling. DNA adduct formation by both alkaloids was found to be concentration dependent. When analyzing different atomic and bond indices of the C11-C12 bond (ring B) in alkaloid molecules we found that fagaronine behaved differently from sanguinarine and chelerythrine. While sanguinarine and chelerythrine showed a preference for electrophilic attack indicating higher potential to be activated by cytochrome P450, fagaronine exhibited a tendency for nucleophilic attack. Our results demonstrate that sanguinarine and chelerythrine are metabolized by hepatic microsomes to species, which generate DNA adducts.     

Conventional protein kinase C isoenzymes undergo dephosphorylation in neutrophil-like HL-60 cells treated by chelerythrine or sanguinarine

The quaternary benzo[c]phenanthridine alkaloid chelerythrine is widely used as an inhibitor of protein kinase C (PKC). However, in biological systems chelerythrine interacts with an array of proteins. In this study, we examined the effects of chelerythrine and sanguinarine on conventional PKCs (cPKCs) and PKC upstream kinase, phosphoinositide-dependent protein kinase 1 (PDK1), under complete inhibition conditions of PKC-dependent oxidative burst. In neutrophil-like HL-60 cells, sanguinarine and chelerythrine inhibited N-formyl-Met-Leu-Phe, phorbol 12-myristate 13-acetate (PMA)-, and A23187-induced oxidative burst with IC₅₀ values not exceeding 4.6 μmol/L, but the inhibition of PMA-stimulated cPKC activity in intact cells required at least fivefold higher alkaloid concentrations. At concentrations below 10 μmol/L, sanguinarine and chelerythrine prevented phosphorylation of ∼80 kDa protein and sequestered ∼60 kDa phosphoprotein in cytosol. Moreover, neither sanguinarine nor chelerythrine impaired PMA-stimulated translocation of autophosphorylated PKCα/βII isoenzymes, but both alkaloids induced dephosphorylation of the turn motif in PKCα/βII. The dephosphorylation did not occur in unstimulated cells and it was not accompanied by PKC degradation. Furthermore, cell treatment with sanguinarine or chelerythrine resulted in phosphorylation of ∼70 kDa protein by PDK1. We conclude that PKC-dependent cellular events are affected by chelerythrine primarily by multiple protein interactions rather than by inhibition of PKC activity.     

Selective inhibition of monoamine oxidase A by chelerythrine,an isoquinoline alkaloid

Chelerythrine, an isoquinoline alkaloid isolated from the herbaceous perennial Chelidonium majus, was found to potently and selectively inhibit an isoform of recombinant human monoamine oxidase-A (MAO-A) with an IC₅₀ value of 0.55?µM. Chelerythrine was a reversible competitive MAO-A inhibitor (K_i?=?0.22?µM) with a potency much greater than toloxatone (IC₅₀?=?1.10?µM), a marketed drug. Other isoquinoline alkaloids tested did not effectively inhibit MAO-A or MAO-B. A structural comparison with corynoline suggested the 1- and/or 2-methoxy groups of chelerythrine increase its inhibitory activity against MAO-A. Molecular docking simulations revealed that the binding affinity of chelerythrine for MAO-A (?9.7?kcal/mol) was greater than that for MAO-B (?4.6?kcal/mol). Docking simulation implied that Cys323 and Tyr444 of MAO-A are key residues for hydrogen-bond interaction with chelerythrine. Our findings suggest chelerythrine is one of the most reversible selective and potent natural inhibitor of MAO-A, and that it be regarded a potential lead compound for the design of novel reversible MAO-A inhibitors.     

不同生境白屈菜(Chelidonium majus)生活史型特征及其与不同器官单宁、黄酮、生物碱含量的关系

运用主成分分析法,对不同生境条件下白屈菜植物生活史型相关的营养生长（vegetative growth,V）、有性生长（sexual growth,S）和克隆生长（clonal growth,C）的主成分得分及比例进行定量计算,并以此为基础研究了生活史型特征参数及次生代谢产物单宁含量、黄酮含量与生物碱含量的相关关系,目的是为植物活性成分环境定向诱导培育提供实验依据。研究结果表明：（1）对于不同光照状况的全光照（空地）、70%光照（榆树林内）和40%光照下（白扦林内）的9个样地白屈菜生活史型划分发现,全光照下白屈菜种群生活史型为V0.34S0.41C0.25,为SV生活史型,空地为DE（Disturbed but still Excellent）生境;榆树林下和白扦林下的白屈菜生活史型分别可表示为V0.28S0.38C0.34和V0.27S0.40C0.33,均为SC生活史型,榆树林下和白扦林下为DF（Disturbed and Fragile）生境。（2）白屈菜植株次生代谢产物（单宁、黄酮和生物碱）含量,空地均低于榆树林下和白扦林下生境。各样地白屈菜不同器官单宁含量：叶片（种（根（茎;黄酮含量：种（叶片（根（茎;生物碱含量：叶片（根（茎,各样地白屈菜茎和根的生物碱含量无明显差异,空地白屈菜叶片中生物碱含量低于榆树林下和白扦林下白屈菜植株59%～56.7%。（3）白屈菜生活史型与次生代谢产物（单宁、黄酮和生物碱）含量相关性分析结果中,显著的线性关系显示,白屈菜次生代谢产物（单宁、黄酮和生物碱）含量与营养生长和有性生长成负相关,与克隆生长成正相关。实验结果表明,较于空地的DE生境,林（榆树和白扦）下的DF生境条件差,使白屈菜向C型转变,同时也促进了次生代谢产物（单宁、黄酮和生物碱）的积累。结果可以为野生植物的人工定向培育中生境选择和目的活性成分定向累积提供基于形态学的评价方法和理论。