Benzophenanthridine Alkaloids Research Articles

Systematic identification of alkaloids in Macleaya microcarpa fruits by liquid chromatography tandem mass spectrometry combined with the isoquinoline alkaloids biosynthetic pathway

Alkaloids in Macleaya microcarpa were characterized systematically by combining liquid chromatography tandem mass spectrometry (LC–MS/MS) with the biosynthetic pathway of isoquinoline alkaloids. The mass spectral fragmentation behaviors of 16 references belonging to eight types of alkaloids that exist in the biosynthetic pathway of isoquinoline were investigated in detail. The benzyltetrahydroisoquinoline and aporphine alkaloids were distinguished by characteristic losses of the NHR1R2 (R1 and R2 represent the substituent groups of the nitrogen atom) radical and the fragment ions below m/z 200. Tetrahydroprotoberberine, N-methyltetrahydroberberine and protopine alkaloids were differentiated by the retro-Diels–Alder (RDA) reaction, α-cleavage and the [M–H2O]+ and [M–CH4]+ ions. Discrimination of protoberberine, benzophenanthridine and dihydrobenzophenanthridine-type alkaloids can be realized through the characteristic [fragment ion-2H]+, [M–H2O]+, [M–CH4]+, [M+H–CH3CH2CH2OH]+ and [M+H–CH3COCH3]+ ions. Forty-one alkaloids, including one benzyltetrahydroisoquinoline, one aporphine, nine protopines, seven protoberberines, one tetrahydroprotoberberine, three N-methyltetrahydroprotoberberines, five benzophenanthridines and fourteen dihydrobenzophenanthridines, were separated and identified simultaneously. Thirty-three of these were reported for the first time in M. microcarpa. The benzyltetrahydroisoquinoline, aporphine, tetrahydroprotoberberine and N-methyltetrahydroprotoberberine-type alkaloids have not been reported previously in M. microcarpa. This method can be applied to the analysis of herbal medicines that possess the biosynthetic pathway of isoquinoline alkaloids.

In vitro cytotoxic activity of alkaloid-containing plant extracts

Alkaloid-containing plant extracts were evaluated for their potential use in the treatment of cancer. Chelidonium majus L. (Papaveraceae), containing benzophenanthridine alkaloids such as chelidonine, sanguinarine, chelerythrine and Gloriosa superba L. (Liliaceae), containing colchicine, 3-O-demethylcolchicine and colchicoside, were selected based on traditional medicine and previously reported cytotoxic activity of their constituents. The cytotoxicity of the 80% ethanolic extract of the C. majus herb and of the G. superba seeds was evaluated in vitro on cell lines of different origin. The effect on pancreatic carcinoma (PANC-1), murine pancreatic carcinoma (PANC02), breast adenocarcinoma (MDA-MB-231) and colorectal adenocarcinoma (HT-29) cell lines was tested with two different incubation times (24h and 72h) using the sulforhodamine B assay. The effect on two primary endometrial cancer cell lines (PC-EM005 and PC-EM002) and on mouse fibroblast cells (3T3) was evaluated with the use of the impedance-based xCELLigence REAL TIME Cell Analysis platform. The IC50 values found for both extracts on the different cell lines are summarized in the table. Plant extracts with an IC50 value lower than 20 µg/mL were considered having a high inhibitory effect and values between 20 – 100 µg/mL having a moderate effect on the tested cell lines. Based upon these results, both ethanolic extracts exhibited promising cytotoxic activity on certain cell lines.

Benzophenanthridine alkaloid macarpine – biological effects and plant sources

Plants with quaternary benzophenanthridine alkaloids (QBAs) are used for a long time in traditional Chinese medicine for analgesic, anti-inflammatory, antiedemic, depurative or diuretic properties. Wide range of effects of these plants had increased the number of studies in the biochemistry or pharmacology in recent years. A great deal of attention is devoted to the main alkaloids sanguinarine and chelerythrine. Besides these commercially available and well-known alkaloids, there are also minor alkaloids such as macarpine (MA) with limited information about its properties in literature. Among the rare QBAs, MA was reported as a promising fluorescent probe for cell nuclei labelling as well as for cell cycle visualisation in fluorescence microscopy and flow cytometry. We have previously described strong antiproliferative effects of MA on several cancer cell lines, but this is the first documentation of their effect on malignant melanoma cells. Taking into account all the biological activities of quaternary benzophenanthridine alkaloids highlight their further application in cancer biology and therapy studies and justify the continuation in the research of MA that is dependent on the isolation of alkaloids from plants sources.

Cytotoxic Benzophenanthridine and Furoquinoline Alkaloids from Zanthoxylum buesgenii (Rutaceae).

BackgroundZanthoxylum buesgenii is a shrub used in Sierra Leone as remedy to cure venereal diseases, arthritis, and rheumatism whereas leaves and barks are employed to treat leprosy and to relieve pain. In South West Region of Cameroon, the plant locally called “Mbem” by Lewoh-Lebang community, is orally given to patients as aphrodisiac decoction and to increase sperm count. Previous chemical studies on Zanthoxylum species reported the identification of lignans, coumarins, diterpenes, sesquiterpenes, steroids, alkaloids and benzopropanoids. Besides, structurally diverse compounds belonging to these classes of secondary metabolites have been reported as trypanocidal, antileishmanial, antimycobacterial and cytotoxic metabolites.ResultsWe therefore investigated the alkaloidal constituents of Z. buesgenii. In the course of the study, two benzophenanthridines [1-methoxy-12-methyl-12,13-dihydro-[1,3]dioxolo[4′,5′:4,5]benzo[1,2-c]phenanthridine-2,13-diol (1) and isofagaridine (2)] were identified among them one new. Alongside, three known furoquinolines [maculine (3), kokusaginine (4) and teclearverdoornine (5)] were also obtained and their structures were established on the basis of their NMR data and by comparison with those previously reported. Furthermore, the cytotoxicities of metabolites (1–4) isolated in substantial amount were evaluated against a series of multidrugs-resistant cancer cell lines. While compounds 2–4 showed selective cytotoxicities, compound 1 displayed activities against all cancer cells.ConclusionsThe observed activities corroborate those previously reported on similar benzophenanthridine alkaloids indicating that compounds 1 and 2 can chemically be explored to develop other chemotherapeutic agents.Graphical abstractCytotoxic Benzophenanthridine and Furoquinoline Alkaloids from Zanthoxylum buesgenii (Rutaceae).Electronic supplementary materialThe online version of this article (doi:10.1186/s13065-014-0061-4) contains supplementary material, which is available to authorized users.

Interaction between Na-K-ATPase and Bcl-2 proteins BclXL and Bak.

In silico analysis predicts interaction between Na-K-ATPase (NKA) and Bcl-2 protein canonical BH3- and BH1-like motifs, consistent with NKA inhibition by the benzo-phenanthridine alkaloid chelerythrine, a BH3 mimetic, in fetal human lens epithelial cells (FHLCs) (Lauf PK, Heiny J, Meller J, Lepera MA, Koikov L, Alter GM, Brown TL, Adragna NC. Cell Physiol Biochem 31: 257-276, 2013). This report establishes proof of concept: coimmunoprecipitation and immunocolocalization showed unequivocal and direct physical interaction between NKA and Bcl-2 proteins. Specifically, NKA antibodies (ABs) coimmunoprecipitated BclXL (B-cell lymphoma extra large) and BAK (Bcl-2 antagonist killer) proteins in FHLCs and A549 lung cancer cells. In contrast, both anti-Bcl-2 ABs failed to pull down NKA. Notably, the molecular mass of BAK1 proteins pulled down by NKA and BclXL ABs appeared to be some 4-kDa larger than found in input monomers. In silico analysis predicts these higher molecular mass BAK1 proteins as alternative splicing variants, encoding 42 amino acid (aa) larger proteins than the known 211-aa long canonical BAK1 protein. These BAK1 variants may constitute a pool separate from that forming mitochondrial pores by specifically interacting with NKA and BclXL proteins. We propose a NKA-Bcl-2 protein ternary complex supporting our hypothesis for a special sensor role of NKA in Bcl-2 protein control of cell survival and apoptosis.

AGS 인체 위암세포에서 DR5의 발현 및 ROS 생성의 증가를 통한 sanguinarine과 TRAIL 혼합처리의 apoptosis 유도 활성 촉진

혈근초(Sanguinaria canadensis) 뿌리에서 유래된 benzophenanthridine alkaloid의 일종인 sanguinarine은 항균, 항산화 및 항암작용 등 다양한 생리활성을 지니고 있는 것으로 알려져 있다. 비록 TRAIL이 암세포에서는 apoptosis를 유도하지만 정상세포에서는 세포독성을 나타내지 않는다는 큰 장점으로 제 2 임상 단계에서 유의적인 성과를 이루었지만, TRAIL 저항성을 극복해야 하는 큰 어려움이 남아있다. 본 연구실에서는 TRAIL이 세포독성을 나타내지 않는 범위의 sanguinarine과 혼합처리에 의하여 TRAIL 저항성 AGS 위암세포에서 apoptosis를 유발하였음을 보고한 바 있으며, 본 연구에서는 sanguinarine의 TRAIL 저항성 관련 극복에 대한 추가적인 기전 연구를 실시하였다. 본 연구의 결과에 의하면, sanguinarine과 TRAIL의 혼합처리는 각각의 단독 처리에 비하여 AGS 세포의 증식억제 및 apoptosis 유도의 상승 효과가 있었으며, 이를 MTT assay, agarode gel 전기영동, 염색질 응축 현상 및 flow cytometry 분석을 통하여 확인하였다. 또한 sanguinarine과 TRAIL의 혼합처리는 DR5의 발현을 증가시켰으며, ROS의 생성을 촉진시켰다. 그러나 동일 조건에서 MAPKs 신호 전달계에는 큰 영향을 주지 않았다. 아울러 ROS 생성을 인위적으로 차단하였을 경우, sanguinarine과 TRAIL의 혼합처리에 의한 생존도 저하가 유의적으로 회복되었다. 이러한 결과는 sanguinarine과 TRAIL이 DR5의 발현 증가와 ROS의 생성을 촉진시킴으로서 apoptosis 신호를 활성화하였음을 의미하는 결과로서 TRAIL 저항성 극복을 위한 sanguinarine 활용의 유용성을 보여주는 것이다. Sanguinarine, a benzophenanthridine alkaloid originally derived from the root of Sanguinaria canadensis, has been shown to possess antimicrobial, antioxidant, and anti-cancer properties. Although tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is known to induce apoptosis in cancer cells, but not most normal cells and has shown efficacy in a phase 2 clinical trial, development of resistance to TRAIL by tumor cells is a major roadblock. Our previous study indicated that treatment with TRAIL in combination with subtoxic concentrations of sanguinarine sensitized TRAIL-mediated apoptosis in TRAIL-resistant human gastric carcinoma AGS cells; however, the detailed mechanisms are not fully understood. In this study, we show that sanguinarine sensitizes AGS cells to TRAIL-mediated apoptosis as detected by MTT assay, agarose gel electrophoresis, chromatin condensation and flow cytometry analysis. Combined treatment with sanguinarine and TRAIL effectively induced expression of death receptor (DR) 5 but did not affect expression of DR4 and mitogen activated protein kinases signaling molecules. Moreover, the combined treatment with sanguinarine and TRAIL increased the generation of reactive oxygen species (ROS); however, N-acetylcysteine, ROS scavenger, significantly recovered growth inhibition induced by the combined treatment. Taken together, our results indicate that sanguinarine can potentiate TRAIL-mediated apoptosis through upregulation of DR5 expression and ROS generation.

Nitidine chloride inhibits hepatic cancer growth via modulation of multiple signaling pathways.

BackgroundThe development of hepatic cancer is tightly regulated by multiple intracellular signaling pathways. Therefore, most currently-used anti-tumor agents, which typically target single intracellular pathway, might not always be therapeutically effective. Additionally, long-term use of these agents probably generates drug resistance and unacceptable adverse effects. These problems increase the necessity for the development of new chemotherapeutic approaches. Nitidine chloride (NC), a natural benzophenanthridine alkaloid, has been shown to inhibit cancer growth via induction of cell apoptosis and suppression of cancer angiogenesis. But the precise mechanisms of its tumorcidal activity are not well understood.MethodsTo further elucidate the precise mechanisms of its anti-tumor activity, using a hepatic cancer mouse xenograft model, the human hepatic cancer cell lines (HepG2, HCCLM3, Huh7), and umbilical vein endothelial cells (HUVEC), here we evaluate the effect of NC on tumor growth in vivo and in vitro and investigated the underlying molecular mechanisms.ResultsWe found that NC treatment resulted in significant decrease in tumor volume and tumor weight respectively, but didn’t affect body weight changes. Additionally, NC treatment dose- and time-dependently reduced the cell viability of all three hepatic cell lines. Moreover, NC suppressed the activation of STAT3, ERK and SHH pathways; and altered the expression of critical target genes including Bcl-2, Bax, Cyclin D1, CDK4, VEGF-A and VEGFR2. These molecular effects resulted in the promotion of apoptosis, inhibition of cell proliferation and tumor angiogenesis.ConclusionsOur findings suggest that NC possesses a broad range of anti-cancer activities due to its ability to affect multiple intracellular targets, suggesting that NC could be a novel multi-potent therapeutic agent for the treatment of hepatic cancer and other cancers.

6-Acetonyldihydrochelerythrine Is a Potent Inducer of Apoptosis in HCT116 and SW620 Colon Cancer Cells.

6-Acetonyldihydrochelerythrine (1), a benzophenanthridine alkaloid, isolated from the methanol extract of Zanthoxylum capense, displayed potent cytotoxic activity in human HCT116 and SW620 colon carcinoma cells, to a higher extent than 5-fluorouracil (5-FU), the cornerstone chemotherapeutic agent in colon cancer. Cytotoxicity of 1 was evaluated by MTS, lactate dehydrogenase (LDH), and Guava ViaCount assays. Interestingly, 1 significantly induced cytotoxicity in both cell lines, leading to a significant increase in LDH release, as compared to 5-FU. Further, Guava ViaCount flow cytometry assays demonstrated that 1 significantly increased cell death, as shown by the presence of a significantly higher population of apoptotic cells in both cell lines, as compared to cells exposed to 5-FU. Furthermore, evaluation of nuclear morphology by Hoechst staining of 1-treated HCT116 and SW620 cells confirmed flow cytometry results, demonstrating a marked induction of apoptotic cell death by 1, again to a further extent than that elicited by 5-FU. In addition, immunoblot analysis to ascertain the molecular events triggered by 1 exposure was performed. The results show that 1 exposure reduced the steady-state expression and activation of the pro-survival proteins ERK5 and Akt and increased the steady-state expression of p53 in both HCT116 and SW620 cells. Changes in ERK5 or Akt activation can be ascertained by evaluating the ratio of p-ERK5/ERK5 or p-Akt/Akt. In addition, exposure to 1 reduced expression of XIAP, Bcl-XL, and Bcl-2, while increasing the cleavage of poly(ADP-ribose) polymerase in both cell lines. Collectively, the data indicate that 6-acetonyldihydrochelerythrine (1) is a potent inducer of apoptosis in HCT116 and SW620 cell lines, highlighting its potential relevance in colon cancer.

Self-regulation of phytoalexin production: a non-biosynthetic enzyme controls alkaloid biosynthesis in cultured cells of Eschscholzia californica

Benzophenanthridine alkaloids are strong antimicrobials of Papaveraceae and attractive lead compounds for drug development. The cytotoxicity of these compounds requires the producing plant to limit the pathogen-triggered burst of biosynthesis. Cells of Eschscholzia californica excrete early benzophenanthridines to the cell wall, followed by re-uptake and reduction in the cytoplasm by the detoxifying enzyme sanguinarine reductase. We now discovered that this enzyme is a core component of self-control in alkaloid production. RNAi-based silencing of sanguinarine reductase gave rise to mutants that either show a complete stop of elicitor-triggered alkaloid production or a burst of biosynthesis that severalfold surpasses the wild type level. These unexpected phenotypes reflect impacts of substrate or product of sanguinarine reductase: the substrate, sanguinarine, inhibits phospholipase A2 at the plasma membrane, an initial component of the signal path towards expression of biosynthetic enzymes. The product, dihydrosanguinarine, inhibits enzymes of early biosynthesis, prior to reticuline formation. By tuning these steady states, sanguinarine reductase adjusts the capacity of alkaloid biosynthesis: a minimum activity is sufficient to prevent the blockade of the induction pathway by sanguinarine, while the full activity of the same enzyme causes a limitation of the biosynthetic flow via dihydrosanguinarine.

Sanguinarine suppresses basal-like breast cancer growth through dihydrofolate reductase inhibition

Basal-like breast cancer (BLBC) remains a great challenge because of its clinically aggressive nature and lack of effective targeted therapy. We analyzed the potential anti-neoplastic effects of sanguinarine, a natural benzophenanthridine alkaloid, against BLBC cells. Sanguinarine treatment resulted in a reduction of cell migration, in a dose-dependent inhibition of cell viability and in the induction of cell death by apoptosis in both human (MDA-MB-231 cells) and mouse (A17 cells) in vitro models of BLBC. In vivo experiments demonstrated that oral administration of sanguinarine reduced the development and growth of A17 transplantable tumors in FVB syngeneic mice. Western blotting analysis revealed that suppression of BLBC growth by sanguinarine was correlated with a concurrent upregulation of p27 and downregulation of cyclin D1 and with the inhibition of STAT3 activation. In addition, we identified sanguinarine as a potent inhibitor of dihydrofolate reductase (DHFR), able to impair enzyme activity even in methotrexate resistant MDA-MB-231 cells. These results provide evidence that sanguinarine is a promising anticancer drug for the treatment of BLBC.

Structure-activity relationship of benzophenanthridine alkaloids from Zanthoxylum rhoifolium having antimicrobial activity.

Zanthoxylum rhoifolium (Rutaceae) is a plant alkaloid that grows in South America and has been used in Brazilian traditional medicine for the treatment of different health problems. The present study was designed to evaluate the antimicrobial activity of the steam bark crude methanol extract, fractions, and pure alkaloids of Z. rhoifolium. Its stem bark extracts exhibited a broad spectrum of antimicrobial activity, ranging from 12.5 to 100 µg/mL using bioautography method, and from 125 to 500 µg/mL in the microdilution bioassay. From the dichloromethane basic fraction, three furoquinoline alkaloids (1–3), and nine benzophenanthridine alkaloids (4–12) were isolated and the antimicrobial activity of the benzophenanthridine alkaloids is discussed in terms of structure-activity relationships. The alkaloid with the widest spectrum of activity was chelerythrine (10), followed by avicine (12) and dihydrochelerythrine (4). The minimal inhibitory concentrations of chelerythrine, of 1.50 µg/mL for all bacteria tested, and between 3.12 and 6.25 µg/mL for the yeast tested, show this compound to be a more powerful antimicrobial agent when compared with the other active alkaloids isolated from Z. rhoifolium. To verify the potential importance of the methylenedioxy group (ring A) of these alkaloids, chelerythrine was selected to represent the remainder of the benzophenanthridine alkaloids isolated in this work and was subjected to a demethylation reaction giving derivative 14. Compared to chelerythrine, the derivative (14) was less active against the tested bacteria and fungi. Kinetic measurements of the bacteriolytic activities of chelerythrine against the bacteria Bacillus subtilis (Gram-positive) and Escherichia coli (Gram-negative) were determined by optical density based on real time assay, suggesting that its mechanism of action is not bacteriolytic. The present study did not detect hemolytic effects of chelerythrine on erythrocytes and found a protective effect considering the decrease in TBARS and AOPP (advanced oxidized protein products) levels when compared to the control group.

Synthesis of 5-methyl phenanthridium derivatives: A new class of human DOPA decarboxylase inhibitors

DOPA decarboxylase (DDC) is responsible for the decarboxylation of l-DOPA and related aromatic amino acids and correlates closely with a number of clinical disorders. Sanguinarine, a natural quaternary benzophenanthridine alkaloid (QBA), was reported to be inhibitor of rat DDC and possessed a different inhibitory mechanism. In this study, several natural QBAs were assayed as human DDC inhibitors for the first time. A series of 5-methyl phenanthridium derivatives that contain the basic core structure of QBAs were also synthesized and evaluated as human DDC inhibitors. The title compounds still possessed DDC inhibitory potential. Among the synthesized compounds, 2-hydroxyl-8-methoxy-5-methylphenanthridinium chloride (11k) showed good inhibitory activity with an IC50 value of 0.12mM. Preliminary structure–activity relationship indicated that DDC inhibitory potential of 5-methyl phenanthridium derivatives correlated with the π-electro densities on CN double bond of iminium cation. The hydroxyl group on compound 11k possibly contributed to the formation of hydrogen bond between DDC and the inhibitor.

Structural speculation and identification of alkaloids in Macleaya cordata fruits by high‐performance liquid chromatography/quadrupole‐time‐of‐flight mass spectrometry combined with a screening procedure

Alkaloids with significant therapeutic effects are the main active constituents of Macleaya cordata, which is a perennial herb plant in the Papaveraceae family. A systematic and novel method for speculating and identifying the structures of alkaloids in M. cordata fruits by high-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry (HPLC/Q-TOF-MS) with a screening procedure was reported. Investigation of mass spectral fragmentation of alkaloids was carried out based on the tandem mass spectrometry (MS/MS) data analyses of eight reference substances. The skeletons of alkaloids were determined by their ultraviolet spectra (UV) and MS/MS data. The substituent groups of the alkaloids were acquired through a screening procedure developed in our laboratory and MS/MS data. The substituent linkage sites were deduced by MS/MS fragmentation behavior, as well as biosynthetic pathways of related alkaloids. The structures of 21 alkaloids were speculated in this study, 10 of which were reported for the first time in M. cordata. Furthermore, benzyltetrahydroisoquinoline and N-methyltetrahydroprotoberberine-type alkaloids were discovered, which indirectly proved that the biosynthetic pathways of benzophenanthridine alkaloids reported in Eschscholtzia california existed in M. cordata as well. HPLC/Q-TOF-MS combined with a screening procedure was a systematic and reliable method for speculating and elucidating the structures of alkaloids. This study might be useful for the identification of other compounds in herbal medicines.

Quantitative Determination of Nitidine from Roots and Plant Tissue Culture Extracts of <i>Toddalia asiatica</i> (Linn.) Using HPTLC

Toddalia asiatica Linn. is an important medicinal plant belonging to the family Rutaceae. The plant is well known for its antimalarial activity, which has been attributed to the presence of benzophenanthridine alkaloid nitidine in the roots of plants. A simple, rapid, sensitive, accurate, repeatable and robust HPTLC method has been developed and validated for the quantitative determination of nitidine in the dried roots and plant tissue culture extracts of T. asiatica. Nitidine was estimated at 332 nm by densitometry using Silica gel 60 F254 as stationary phase and chloroform:methanol (7:1, v/v), and as mobile phase. Linearity was observed in the concentration range of 25 -200 ng/spot for nitidine. The limit of detection and limit of quantitation were found to be 0.026 and 0.086 ng/spot respectively for nitidine. Developed method was validated according to the ICH guidelines with respect to precision, accuracy, specificity and robustness. The technique has been applied for the first time for the estimation of nitidine in roots and plant tissue culture extracts of T. asiatica. Statistical analysis data indicate the accuracy and reliability of the method.

Screening of Isoquinoline Alkaloids for Potent Lipid Metabolism Modulation withCaenorhabditis elegans

Metabolic syndrome and related disorders are increasingly prevalent in contemporary society, and thus pose the need for potent agents to control lipid accumulation in the body. This study indicates that Caenorhabditis elegans was effective in screening for potent lipid metabolism modulators with berberine as a model compound. Among the various isoquinoline alkaloids tested, sanguinarine, a benzophenanthridine alkaloid, was found to be the most potent. Sanguinarine, like berberine, reduced lipid accumulation through AMP-activated protein kinase activation. Analysis of AMPK (aak-1 and aak-2) RNAi worms revealed that effects were aak-2-dependent. Characterization of worms with knockdown nhr-49, a hormone nuclear receptor gene that functions as a key regulator of fat consumption, showed that both alkaloids were effective even in these markedly lipid-accumulating nhr-49 RNAi worms, suggesting that they predominantly affect lipid synthesis, rather than fatty acid β-oxidation. The versatility of C. elegans for the purpose of lipid-modulating chemical screening and characterization of the underlying mechanisms is discussed.

Effects of quaternary benzo(c)phenanthridine alkaloids on growth performance, shedding of organisms, and gastrointestinal tract integrity in pigs inoculated with multidrug-resistant Salmonella spp

To evaluate effects of quaternary benzo(c)phenanthridine alkaloids (QBAs) against Salmonella spp and determine effects on growth performance, organism shedding, and gastrointestinal tract integrity in pigs inoculated with Salmonella enterica serovar Typhimurium. 36 Salmonella isolates and twenty 5-week-old pigs. Minimum inhibitory concentration of QBAs against the Salmonella isolates was determined. Pigs were allocated to 4 groups and inoculated with Salmonella organisms. Pigs received diets supplemented with 1.5 g of QBAs/1,000 kg of feed, 0.75 g of QBAs/1,000 kg of feed, or 59.4 g of chlortetracycline/1,000 kg of feed or a nonsupplemented (control) diet. Pigs were weighed on day 0 and then weekly for 40 days. Fecal samples were collected to quantify Salmonella organisms. Gastrointestinal tract integrity was evaluated by measuring transepithelial resistance. In vitro, 9 of 36 (25%) Salmonella isolates were inhibited at 90 μg of QBAs/mL; all 36 were inhibited at 179 μg of QBAs/mL. Diets containing QBAs significantly decreased Salmonella spp shedding; shedding was lower 40 days after inoculation for pigs fed diets containing QBAs or chlortetracycline than for pigs fed the control diet. Growth performance was similar for pigs fed diets containing QBA or chlortetracycline. Gastrointestinal tract integrity was improved in pigs fed the diet containing 1.5 g of QBAs/1,000 kg of feed. QBAs and chlortetracycline decreased Salmonella spp shedding but did not differ with regard to growth performance. Gastrointestinal tract integrity was better, albeit not significantly, in pigs fed diets containing QBAs. Further investigation into the role of QBAs and their mechanism as an immunomodulator is necessary.

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.

Effects of bloodroot (Sanguinaria canadensis L.) rhizome ethanol extracts on cytokine production by blood mononuclear cells during flowering and fruiting

Extracts from bloodroot (Sanguinaria canadensis L., Papaveraceae) are used in herbal medicines due to their anti-bacterial, anti-inflammatory, immunomodulatory, and tumouricidal properties. Benzophenanthridine alkaloids are the purported bioactive molecules. Although substantial investigative attention has been given to isolated alkaloids, little attention has been given to whole-plant extracts such as those used in both traditional and contemporary herbal medicine. The purposes of this investigation were to: (a) investigate the immunomodulatory properties of bloodroot rhizome ethanol extracts using a human peripheral blood mononuclear cell model of in vitro cytokine production and cell proliferation; (b) characterize alkaloid content of the extracts using liquid chromatography–tandem mass spectrometry (LC/MS); and, (c) determine if the flowering and fruiting status of bloodroot influenced the preceding outcomes by sampling plants over a 6-week springtime period. LC/MS detected seven quaternary benzophenanthridine alkaloids, six which could be quantified (chelerythrine, chelilutine, chelirubine, sanguilutine, sanguirubine, and sanguinarine) and one detected in trace amounts (protopine). Sanguinarine was most abundant. Extracts demonstrated immunosuppressive effects on production of the cytokines interleukin (IL)-2 and IL-8, and cell proliferation. Production of other cytokines including IL-1β, IL-4, IL-6, IL-10, and tumour necrosis factor was not influenced by treatment. Extract immunomodulatory activities were consistent across all time points sampled. Alkaloid content did not differ significantly across the time points sampled and could not be correlated to immune outcomes, suggesting that traditional marker components may not adequately explain bloodroot rhizome extract immunomodulatory activity.

Benzophenanthridine alkaloid, piperonyl butoxide and (S)-methoprene action at the cannabinoid-1 receptor (CB1-receptor) pathway of mouse brain: Interference with [3H]CP55940 and [3H]SR141716A binding and modification of WIN55212-2-dependent inhibition of synaptosomal l-glutamate release

Benzophenanthridine alkaloids (chelerythrine and sanguinarine) inhibited binding of [3H]SR141716A to mouse brain membranes (IC50s: <1µM). Piperonyl butoxide and (S)-methoprene were less potent (IC50s: 21 and 63µM respectively). Benzophenanthridines and piperonyl butoxide were more selective towards brain CB1 receptors versus spleen CB2 receptors. All compounds reduced Bmax of [3H]SR141716A binding to CB1 receptors, but only methoprene and piperonyl butoxide increased Kd (3–5-fold). Benzophenanthridines increased the Kd of [3H]CP55940 binding (6-fold), but did not alter Bmax. (S)-methoprene increased the Kd of [3H]CP55940 binding (by almost 4-fold) and reduced Bmax by 60%. Piperonyl butoxide lowered the Bmax of [3H]CP55940 binding by 50%, but did not influence Kd. All compounds reduced [3H]SR141716A and [3H]CP55940 association with CB1 receptors. Combined with a saturating concentration of SR141716A, only piperonyl butoxide and (S)-methoprene increased dissociation of [3H]SR141716A above that of SR141716A alone. Only piperonyl butoxide increased dissociation of [3H]CP55940 to a level greater than CP55940 alone. Binding results indicate predominantly allosteric components to the study compounds action. 4-Aminopyridine-(4-AP-) evoked release of l-glutamate from synaptosomes was partially inhibited by WIN55212-2, an effect completely neutralized by AM251, (S)-methoprene and piperonyl butoxide. With WIN55212-2 present, benzophenanthridines enhanced 4-AP-evoked l-glutamate release above 4-AP alone. Modulatory patterns of l-glutamate release (with WIN-55212-2 present) align with previous antagonist/inverse agonist profiling based on [35S]GTPγS binding. Although these compounds exhibit lower potencies compared to many classical CB1 receptor inhibitors, they may have potential to modify CB1-receptor-dependent behavioral/physiological outcomes in the whole animal.

The benzophenanthridine alkaloid chelerythrine binds to DNA by intercalation: Photophysical aspects and thermodynamic results of iminium versus alkanolamine interaction

The interaction of the natural benzophenanthridine alkaloid chelerythrine with DNA was studied by spectroscopy, viscometry and calorimetry techniques. The absorbance and fluorescence properties of the alkaloid were remarkably modified upon binding to DNA and the interaction was found to be cooperative. The mode of binding was principally by intercalation as revealed from viscosity studies and supported from fluorescence quenching, and polarization results. The binding remarkably stabilized the DNA structure against thermal strand separation. The binding induced conformational changes in the B-form structure of the DNA and the bound alkaloid molecule acquired induced circular dichroism. The binding affinity values obtained from spectroscopy, fluorescence polarization (and anisotropy) and calorimetry were in agreement with each other. The binding was exothermic, characterized by negative enthalpy and positive entropy change and exhibited enthalpy–entropy compensation phenomenon. The heat capacity changes of the binding revealed hydrophobic contribution to the binding. Molecular aspects of the interaction characterized by the involvement of multiple weak noncovalent forces are presented.