Intracellular Accumulation as an Indicator of Cytotoxicity to Screen Hepatotoxic Components of Chelidonium majus L. by LC-MS/MS.

A novel strategy was developed to identify hepatotoxic compounds in traditional Chinese medicines (TCMs). It is based on the exposure of HL-7702 cells to a TCM extract, followed by the identification and further determination of potential hepatotoxic compounds accumulated in the cells by liquid chromatography-tandem mass spectrometry (LC-MS/MS). As a case study, potential hepatotoxic components in Chelidonium majus L. were screened out. Five alkaloids (sanguinarine, coptisine, chelerythrine, protopine, and chelidonine) were identified by LC-MS/MS within 10 min, and their intracellular concentrations were first simultaneously measured by LC-MS/MS with a run time of 4 min. A cell viability assay was performed to assess the cytotoxicity of each alkaloid. With their higher intracellular concentrations, sanguinarine, coptisine, and chelerythrine were identified as the main hepatotoxic constituents in Ch. majus. The study provides a powerful tool for the fast prediction of cytotoxic components in complex natural mixtures on a high-throughput basis.

Protopine attenuates inflammation stimulated by carrageenan and LPS via the MAPK/NF-κB pathway.

We investigated the anti-inflammatory activity of protopine (PTP) and sought to determine its mechanism of action in LPS-stimulated BV2 cells and a carrageenan (CA)-induced mouse model. Treatment with PTP (5, 10, and 20 µM) significantly suppresses the secretion of NO and PGE2 in a concentration-dependent manner without affecting cell viability by downregulating iNOS and COX-2 expression in LPS-induced BV2 cells. PTP also attenuates the production of pro-inflammatory chemokines, such as MCP-1, and cytokines, including TNF-α, IL-1ß and IL-6, and augments the expression of the anti-inflammatory cytokine IL-10. In addition, PTP suppresses the nuclear translocation of NF-κB by hindering the degradation of IκB and downregulating the expression of mitogen-activated protein kinases (MAPKs), including p38, ERK1/2 and JNK protein. Furthermore, PTP treatment significantly suppresses CA-induced paw oedema in mice compared to that seen in untreated mice. Expression of iNOS and COX-2 proteins is also abrogated by PTP (50 mg/kg) treatment in CA-induced mice. PTP treatment also abolishes IκB phosphorylation, which hinders the activation of NF-κB. Collectively, these results suggest PTP has potential for attenuating CA- and LPS-induced inflammatory symptoms through modulation of MAPKs/NF-κB signaling cascades.

Characterization of the cytotoxicity of selected Chelidonium alkaloids in rat hepatocytes.

Phytomedicinal preparations containing extracts of the plant Chelidonium majus (Greater Celandine) have been used in the therapy of upper abdominal disorders. C. majus alkaloids (CAL) were suspected to be responsible for reported cases of liver symptoms including cases of acute liver failure in patients upon treatment with certain C. majus preparations. Based on these reports, a safe oral daily dose limit of not more than 2.5 mg CAL was established in the EU. However, C. majus extracts and individual CAL were not able to elicit similar adverse effects when given orally to pigs or rats. We found that CAL differ considerably in their cytotoxicity in rat hepatocytes in culture. The cationic congeners chelerythrine, coptisine and sanguinarine were the most toxic ones (EC20 values ≤2 µM) while the neutral congeners chelidonine, dihydrosanguinarine and protopine were less toxic, with a rank order of toxicity of coptisine > chelerythrine > sanguinarine > chelidonine > protopine > dihydrosanguinarine. Calculation of octanol-water partition coefficients revealed that the most cytotoxic CAL in hepatocytes were the cationic polar ones. At cytotoxic concentrations sanguinarine led to a marked decrease in reduced and oxidized intracellular glutathione while the much less cytotoxic dihydrosanguinarine did not. After glutathione depletion with menadione, CAL toxicity was only slightly enhanced. Comparison of the cytotoxic concentrations to reported liver levels in experimental animals suggests that the latter were too low to cause hepatotoxicity, probably due to an extremely low oral availability of certain CAL.

Assessing the risk of epidemic dropsy from black salve use.

Epidemic dropsy is a potentially life-threatening condition resulting from the ingestion of argemone oil derived from the seeds of Argemone mexicana Linn. Exposure to argemone oil is usually inadvertent, arising from mustard cooking oil adulteration. Sanguinarine, an alkaloid present in argemone oil, has been postulated as a causative agent with the severity of epidemic dropsy correlating with plasma sanguinarine levels. Cases of epidemic dropsy have also been reported following the topical application of argemone containing massage oil. Black salve, a topical skin cancer therapy also contains sanguinarine, but at significantly higher concentrations than that reported for contaminated massage oil. Although not reported to date, a theoretical risk therefore exists of black salve inducing epidemic dropsy. This literature review explores the presentation and pathophysiology of epidemic dropsy and assesses the risk of it being induced by black salve.

Supramolecular formulation of nitidine chloride can alleviate its hepatotoxicity and improve its anticancer activity.

Nitidine chloride (NC) has demonstrated promising anticancer activity. However, NC has also shown non-specific toxicity in various healthy organs such as the liver. In this study, we aimed to develop a supramolecular formulation of NC and investigate the associated benefits of such a supramolecular formulation on modulating its inherent hepatotoxicity and anticancer activity. The formation of NC-cucurbit[7]uil (NC@CB[7]) complexes was characterized by 1H nuclear magnetic resonance and Fourier transform infrared spectroscopy, differential scanning calorimetry and powder X-ray diffraction analysis. As a consequence of the supramolecular complexation, NC@CB[7] showed significantly lower toxicity (IC50: 6.87 ± 0.80 µM) on a liver cell line (LO2), and higher cytotoxicity (IC50: 2.94 ± 0.15 µM) on a breast cancer cell line (MCF-7), when compared with the free drug (IC50 of 3.48 ± 0.49 µM and 7.28 ± 0.36 µM, on these two cell lines, respectively). Investigation of cellular uptakes revealed that CB[7]'s capability in modulating the toxicity/activity of NC was mainly attributed to the drug's different cellular uptake behaviors that were influenced by CB[7]'s complexation. Taken together, we have demonstrated that supramolecular formulation of NC by CB[7] significantly alleviated its hepatotoxicity and improved its anticancer activity in vitro.

Isolation and characterization of the alkaloid Nitidine responsible for the traditional use of Phyllanthus muellerianus (Kuntze) Excell stem bark against bacterial infections.

Phyllanthus muellerianus (Kuntze) Excell (family Euphorbiaceae) stem bark methanol extract inhibited the growth of Clostridium sporogenes and Streptococcus pyogenes, responsible for gas gangrene and suppurative and non suppurative diseases, respectively. After the HPLC fingerprint acquisition a bioguided fractionation of the defatted methanol extract allowed the isolation of six fractions whose activity was evaluated against the two pathogen bacteria. A further purification of the most active fraction afforded a pure compound responsible for the very interesting inhibitory activity against C. sporogenes and S. pyogenes (MIC 0.91 µM, MIC 3.64 µM). (1)H NMR and MS analytical techniques allowed the identification of the bioactive as Nitidine; this quaternary ammonium alkaloid was observed in the genus Phyllanthus for the first time. A study on Nitidine counter ion, performed using energy dispersive spectroscopy (EDS) coupled with scanning electron microscopy (SEM) was also carried out.

Laboratory investigation of epidemic dropsy in Addis Ababa, Ethiopia.

BACKGROUND: Food adulteration including adulteration of edible oils may cause serious health problems. One of the most common edible adulterants is argemone oil. An outbreak of epidemic dropsy occurred in Addis Ababa during May-June, 2008. One hundred and eighty two cases were recorded with twelve confirmed deaths. Dietary history of the cases revealed that vegetable oils were the usual cooking medium. OBJECTIVE: The aim of the study was hence to investigate the causes of this outbreak. METHODS: Contaminant identification was done using standard chemical tests, complemented with TLC. Toxicity study was done using Swiss albino mice feed with contaminated and non contaminated standard diet for 30 days. RESULTS: Laboratory investigation of the edible oils has indicated that 47 of the 280 edible oils analyzed were adulterated with argemone oil. About 81% of the edible oil samples collected from Lideta sub-city were adulterated with argemone oil. Toxicological investigation of the adulterated oils also indicated typical features of argemone alkaloid poisoning in mice. CONCLUSION: Results of both laboratory analysis and toxicological studies confirmed consumption of edible oils adulterated with argemone oil as the cause of epidemic dropsy in Addis Ababa.

Potentiation of tumour promotion by topical application of argemone oil/isolated sanguinarine alkaloid in a model of mouse skin carcinogenesis.

Several incidences of adverse effects on human health have been reported in many countries, due to consumption of edible oil adulterated with argemone oil (AO). The clinical manifestation of the disease is commonly referred to as epidemic dropsy. Our prior studies have shown that AO and isolated sanguinarine alkaloid (SANG) possess genotoxic and tumour initiating activity. In this study, the effect of AO/SANG was investigated on the development of tumour formation in mice using 7,12-dimethylbenz (a) anthracene (DMBA) initiated followed by tetradecanoyl phorbol acetate (TPA)-promoted skin tumour protocol. Single application of AO (300µl) or SANG (4.5µmol) when used during initiation phase in DMBA/TPA group did not reveal substantial difference in tumourigenic response. However, twice weekly application of AO (100µl) or SANG (1.5µmol) during promotion phase (25 weeks) resulted in enhanced tumourigenic response by ≥30% in DMBA/TPA treated group along with significant decrease in dermal tyrosinase (45-49%), histidase (30-32%), superoxide dismutase (53-56%), catalase (41%), GSH reductase (37-40%) and GSH-peroxidase activity (29-33%) compared to control. Furthermore, significant decrease of epidermal GSH (64-66%) content and enhanced formation of lipid peroxides (96-121%) was noticed following AO or SANG treatment during promotion phase to DMBA/TPA induced animals indicating the modified pro-oxidant status in skin. Although dermal biochemical parameters were also altered by AO or SANG when used during initiation phase in DMBA/TPA treated animals, nonetheless, the response in these parameters were relatively more when AO or SANG were used during promotion phase in DMBA/TPA treated animals. These results clearly suggest that AO and SANG have the ability to enhance the tumourigenic response, which may have relevance to its carcinogenic potential.

Phenotype of hepatic xenobiotic metabolizing enzymes and CYP450 isoforms of sanguinarine treated rats: effect of P450 inducers on its toxicity.

Catalytic and immunochemical activities of cytochrome P450 (CYP) isoforms were investigated in argemone alkaloid, sanguinarine (SAN) intoxicated rats, pre-treated with different CYP inducers. SAN treated control (CON) and ethanol (ET), 3- methylcholantherene (MC) or dexamethasone (DEX) pre-exposed rats, resulted in 48, 64, 47 and 33% decrease in CYP content. SAN exposure to CON, and DEX, MC or ET pre-treated animals caused a decrease (22-37%) in glutathione-S-transferase (GST) activity, however, quinone reductase (QR) activity decreased (26-45%) in the MC pre-exposed group. Similarly, western-blot analysis of hepatic CYP1A1 and CYP1A2 showed a decrease (27-37%) in MC pre-treated SAN exposed animals. Further, a decrease in mortality in the SAN+MC (25%) group compared to SAN treated animals was also observed. The results suggest that inhibition of CYP 1A1, 1A2, 2D1, 2E1, 3A1, and Phase II enzymes by SAN augments its toxicity, whereas attenuation of SAN toxicity by MC may be due to removal of parent compound/metabolites from the body.

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.

Sanguinarine.

Sanguinarine is an alkaloid found in many medicinal plants. It has diverse biological activities, including modulation of nuclear factor-kappaB and of several enzymes. It is also known to induce apoptosis, perturb microtubules, and to have antimicrobial effects. This article reviews its cardiovascular properties, including hypotensive, antiplatelet, and positive inotropic effects. Its pharmacokinetics, and toxicology, including its carcinogenic potential, are also discussed. Further pharmacological and toxicological studies with sanguinarine are needed before its therapeutic use can be considered.

Protective effect of bioantioxidants on argemone oil/sanguinarine alkaloid induced genotoxicity in mice.

Our prior studies have shown that argemone oil (AO) and its alkaloid sanguinarine causes DNA damage in mice and Epidemic Dropsy patients. Since some of the bioantioxidants including riboflavin and alpha-tocopherol offered protection to Epidemic Dropsy patients, a combination of riboflavin and alpha-tocopherol was evaluated on AO and sanguinarine induced genotoxicity using alkaline comet assay. Single administration of combination of riboflavin (50mg/kg) and alpha-tocopherol (150mg/kg) to mice, 24h prior to or immediately after AO (2.0ml/kg) exposure showed significant decrease in tail moment (70-72%), tail length (37-44%), and tail DNA (49-53%) in bone marrow cells. Single or multiple doses of antioxidants given after 24h of AO exposure resulted in substantial (P<0.05) decrease in all the parameters of comet assay in bone marrow cells. Single dose of antioxidants given either 24h prior to or immediately after sanguinarine (21.6mg/kg) exposure caused significant decrease in tail moment (56-62%), tail length (69%) and tail DNA (34-42%) in bone marrow cells of mice. Single or multiple doses of antioxidants given after 24h of sanguinarine treated resulted in decrease in tail moment (50-71%), tail length (54-63%) and tail DNA (29-43%) in bone marrow cells. Similar protective response of combination of antioxidants was observed in blood cells of mice treated either with AO or sanguinarine alkaloid. Further, the frequency of bone marrow and blood cells in Olive tail moment category of 8 and onwards were found to be substantially reduced in antioxidants treated animals as compared to respective AO or sanguinarine exposed mice. Based on these results, it can be suggested that a combination of riboflavin and alpha-tocopherol provides protection against AO and sanguinarine induced genotoxicity.