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.     

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.     

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.     

Correlation of the cytotoxic activity of four different alkaloids, from Chelidonium majus (greater celandine), with their DNA intercalating properties and ability to induce breaks in the DNA of NK/Ly murine lymphoma cells

The purpose of this study was to examine the relationship between the DNA intercalating characteristics and the DNA damaging capacity of four alkaloids extracted from Chelidonium majus L, as well as their toxicity towards murine NK/Ly lymphoma cells. Chelerythrine, sanguinarine and coptisine were found to be intercalated into the DNA isolated from NK/Ly cells, meanwhile, chelidonine exhibited no affinity to DNA. Sanguinarine exhibited the greatest toxicity toward NK/Ly cells, and the toxicity of the other three decreased in descending order: chelerythrine, coptisine and chelidonine. Chelerythrine and sanguinarine caused DNA damage, illustrated by the formation of comets of the third class. Coptisine was less toxic than chelerythrine and sanguinarine, and affected the formation the same class of comets in higher concentration. The quantity of comets induced by chelidonine were negligible, a finding consistent with its inability to intercalate into DNA structure. The ability of four main alkaloids of Chelidonium majus L., to intercalate into DNA isolated from murine NK/Ly lymphoma cells, correlated with their ability to induce breaks in cellular DNA and with their toxic effect towards those cells.     

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.     

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.     

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.     

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.     

Rapid determination of protopine, allocryptopine, sanguinarine and chelerythrine in fruits of Macleaya cordata by microwave-assisted solvent extraction and HPLC-ESI/MS

An isocratic high-performance liquid chromatographic method coupled with electrospray mass spectrometry was developed to determine protopine, allocryptopine, sanguinarine and chelerythrine in fruits of Macleaya cordata. The sample was extracted with hydrochloric acid aqueous solution using microwave-assisted extraction method. The extracts were separated on a C8 reversed-phase HPLC column with acetonitrile:acetate buffer as mobile phase, and full elution of all analytes was realized isocratically within 10 min. The abundance of pseudomolecule ions was recorded using selected ion recording at m/z 354.4, 370.1, 332.5, 348.5 and 338.5 for protopine, allocryptopine, sanguinarine, chelerythrine and the internal standard, jatrorrhizine, respectively. Internal standard curves were used for the quantification of protopine, allocryptopine, sanguinarine and chelerythrine, which showed a linear range of 0.745-74.5, 0.610-61.0, 0.525-105 and 0.375-75 microg/mL, respectively, with correlation coefficients of 0.9995, 0.9992, 0.9993 and 0.9989, and limits of detection of 3.73, 3.05, 1.60 and 1.11 ng/mL, respectively.     

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.     

Macleaya cordata extract and Sangrovit genotoxicity. Assessment in vivo

Background: Sanguinarine (SG) has been reported to form DNA adducts in vitro and increase the levels of DNA single strand breaks in the blood and bone marrow of mice treated intraperitoneally with SG. Recently, we showed no genotoxic effects of orally administrated 120 mg/kg feed Macleaya cordata extract (a mixture of sanguinarine and chelerythrine) in pigs or rats in 90-day studies. The goal of this paper was to assess the possible genotoxicity of M. cordata extract when included as a dietary admixture to rodents at concentrations providing 600 mg/kg feed and 100, 7000 or 14000 mg/kg feed Sangrovit (natural feed additive containing M. cordata extract and powdered M. cordata) in a 90-day pilot study. Methods and Results: The rats consumed ad libitum either the standard diet or the diets containing 367 ppm of sanguinarine and chelerythrine in M. cordata extract, and 5, 330, or 660 ppm of total alkaloids in Sangrovit for 90 days. The DNA adducts formation in liver was analyzed by (32)P-postlabeling technique and DNA single strand breaks in lymphocytes were evaluated by Comet assay. The results showed that M. cordata extract and/or Sangrovit induced no DNA damage to rat lymphocytes or hepatocytes after 90-days oral administration. Conclusions: Data from the studies described in this paper and the fact that Sangrovit given to the rats in our experiments were higher than the recommended dose (50 to 100 mg/kg feed), argue strongly in favour of the use of Sangrovit in live stock.     

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.     

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。     

Phylogenetic relationships of Coreanomecon (Papaveraceae: Chelidonioideae) inferred from seed morphology and nrITS sequence data

The phylogenetic position of the Coreanomecon, a monotypic genus endemic to Korea, has been controversial for a long time. Coreanomecon has variously been placed in its own genus or combined with Hylomecon or Chelidonium. The main purpose of this study was to examine the phylogenetic position of Coreanomecon in relation to genera of the subfamily Chelidonioideae using seed morphology and molecular data. The seed morphology of 10 genera of Chelidonioideae was examined using microtome sections and scanning electron microscopy. The shape and size of exotestal cells varied between genera. The exotestal cells were tangentially elongated in Chelidonium majus, Stylophorum diphyllum and Hylomecon vernalis, whereas the cells were sub‐orbicular and broad in Coreanomecon hylomeconoides. The endotesta was represented by thick palisade‐like cells that contain small rectangular crystals near their outer walls. The mesotesta was totally collapsed in Co. hylomeconoides but wholly or partly persistent in Ch. majus and H. vernalis. The seed surface of Co. hylomeconoides was well ornamented with a unique echinate seed surface, whereas other genera showed plain and reticulate seed surfaces. A phylogenetic analysis of the genera including Coreanomecon was conducted with nuclear ribosomal internal transcribed spacer (nrITS) sequences using genetic distance, maximum parsimony, maximum likelihood and Bayesian inference methods. The results confirm that Co. hylomeconoides is separated from both Hylomecon species and is a sister group to Chelidonium majus and Stylophorum diphyllum with robust bootstrap support. In addition to differences in the absence of cauline leaves and rhizomes, the presence of a hairy stem and leaves, and 12‐pericolpate pollen, seed characters and molecular data strongly support the recognition of Coreanomecon as an independent genus, distinct from Hylomecon and Chelidonium.     

Optimizing the Production of Antioxidant and Antibacterial Alkaloids from Macleaya cordata by Subcritical Water Extraction Technology

In this work, the subcritical water extraction technology was used to extract alkaloids from Macleaya cordata, and the effects of extraction temperature and time on its yield were investigated to find the best extraction conditions. Moreover, the antioxidant capacity and antibacterial activity of Macleaya cordata extract were studied. Furthermore, through the single-factor method, it was found that properly increasing the extraction temperature and prolonging the extraction time was conducive to increasing alkaloid yield. Still, a considerable amount of alkaloids might be decomposed by heat, resulting in a decrease in their yield. The results showed that the optimal extraction temperature of alkaloids from Macleaya cordata with subcritical water is 190 °C, the time is 45 min, and the corresponding maximum yield is 35.19±0.12 mg/g (sanguinarine equivalent in raw materials). In addition, the antioxidation and bacteriostasis abilities of subcritical water extract are better than those of traditional hot water extract, indicating that it is a feasible method to extract alkaloids from Macleaya cordata with subcritical water.     

Production of hydrogen peroxide and redox cycling can explain how sanguinarine and chelerythrine induce rapid apoptosis

Sanguinarine and chelerythrine are naturally occurring benzophenanthridines with multiple biological activities. Sanguinarine is believed to be a potential anticancer agent but its mechanism of action has not been fully elucidated. We previously found that it causes oxidative DNA damage and very rapid apoptosis that is not mediated by p53-dependent DNA damage signaling. Here we show that sanguinarine and chelerythrine cause the production of large amounts of reactive oxygen species (ROS), in particular hydrogen peroxide, which may deplete cellular antioxidants and provide a signal for rapid execution of apoptosis. Several oxidoreductases contribute to cell death induced by sanguinarine and chelerythrine which appear to be reduced upon entering the cell. We propose a model in which the generation of lethal amounts of hydrogen peroxide is explained by enzyme-catalyzed redox cycling between the reduced and oxidized forms of the phenanthridines and discuss the implications of such a mechanism for potential pharmaceutical applications.     

Alkaloid metabolism in thrips-Papaveraceae interaction: Recognition and mutual response

Frankliniella occidentalis (Pergande), the Western Flower Thrips (WFT), is a polyphagous and highly adaptable insect of the order Thysanoptera. It has a broad host range but is rarely found on Papaveraceae, which might be due to deterrent effects of alkaloids present in most species of this family. In order to test the adaptive potential of WFT, we investigated its interaction with two Papaveraceae offered as sole feeding source. We found that WFT are able to live and feed on leaves of Eschscholzia californica and Chelidonium majus. Both plants respond to thrips feeding by the enhanced production of benzophenanthridine alkaloids. Furthermore, cell cultures of E. californica react to water insoluble compounds prepared from adult thrips with enhanced alkaloid production. During feeding, WFT take up benzophenanthridine alkaloids from either plant and from an artificial feeding medium and convert them to their less toxic dihydroderivatives. This was shown in detail with sanguinarine, the most cytotoxic benzophenanthridine. A similar conversion is used in plants to prevent self-intoxication by their own toxins. We conclude that WFT causes a phytoalexin-like response in Papaveraceae, but is able to adapt to such host plants by detoxification of toxic alkaloids.     

Biophysical aspects and biological implications of the interaction of benzophenanthridine alkaloids with DNA

Benzophenanthridine alkaloids represent a very interesting and significant group of natural products that exhibit a broad range of biological and pharmacological properties. Among this group of alkaloids, sanguinarine, nitidine, fagaronine, and chelerythrine have the potential to form molecular complexes with DNA structures and have attracted recent attention for their possible clinical and pharmacological utility. This review focuses on the interaction of these alkaloids with polymorphic DNA structures (B-form, Z-form, H^L-form, and triple helical form) reported by several research groups employing various physical techniques such as spectrophotometry, spectrofluorimetry, circular dichroism, NMR spectroscopy, thermal melting, viscometry as well as thermodynamic analysis by isothermal titration calorimetry and differential scanning calorimetry to elucidate the mode and mechanism of action at the molecular level to determine the structure-activity relationship. DNA binding properties of these alkaloids are interpreted in relation to their biological activity.