Novel Approaches for the Analysis and Isolation of Benzylisoquinoline Alkaloids in Chelidonium majus.

Benzylisoquinoline alkaloids are the major bioactive components in Chelidonium majus, a plant that has a long usage history for the treatment of gastrointestinal ailments in European and Asian phytomedicine. This study reports on the development and application of a supercritical fluid chromatography technique for the simultaneous qualitative and quantitative determination of seven benzylisoquinoline alkaloids in under six minutes using a Viridis BEH 2-EP column and a modifier comprising methanol with 30% acetonitrile and 20 mM ammonium formate. The method was fully validated according to ICH guidelines showing, e.g., excellent linearity (≥ 0.9997) and maximum deviations for intraday and inter-day precision of 2.99 and 2.76%, respectively. The new supercritical fluid chromatography assay was not only employed for the analysis of several C. majus samples but was also used for the subsequent development of a fast centrifugal partition chromatography technique, whereby five benzylisoquinoline alkaloids could be isolated within approximately 2.5 h, with only two of them, protopine and chelidonine, requiring an additional purification step. To achieve this, a solvent system composed of chloroform/methanol/0.3 M hydrochloric acid was used in descending mode. By injecting 500 mg of crude extract, stylopine (1.93 mg), sanguinarine (0.57 mg), chelidonine (1.29 mg), protopine (1.95 mg), and coptisine (7.13 mg) could be obtained. The purity of compounds was confirmed by supercritical fluid chromatography and MS.

Identification and Quantification, Metabolism and Pharmacokinetics, Pharmacological Activities, and Botanical Preparations of Protopine: A Review.

Through pharmacological activity research, an increasing number of natural products and their derivatives are being recognized for their therapeutic value. In recent years, studies have been conducted on Corydalis yanhusuo W.T. Wang, a valuable medicinal herb listed in the Chinese Pharmacopoeia. Protopine, one of its components, has also become a research hotspot. To illustrate the identification, metabolism, and broad pharmacological activity of protopine and the botanical preparations containing it for further scientific studies and clinical applications, an in-depth and detailed review of protopine is required. We collected data on the identification and quantification, metabolism and pharmacokinetics, pharmacological activities, and botanical preparations of protopine from 1986 to 2021 from the PubMed database using "protopine" as a keyword. It has been shown that protopine as an active ingredient of many botanical preparations can be rapidly screened and quantified by a large number of methods (such as the LC-ESI-MS/MS and the TLC/GC-MS), and the possible metabolic pathways of protopine in vivo have been proposed. In addition, protopine possesses a wide range of pharmacological activities such as anti-inflammatory, anti-platelet aggregation, anti-cancer, analgesic, vasodilatory, anticholinesterase, anti-addictive, anticonvulsant, antipathogenic, antioxidant, hepatoprotective, neuroprotective, and cytotoxic and anti-proliferative activities. In this paper, the identification and quantification, metabolism and pharmacokinetics, pharmacological activities, and botanical preparations of protopine are reviewed in detail to lay a foundation for further scientific research and clinical applications of protopine.

Cytotoxic and Proapoptotic Activity of Sanguinarine, Berberine, and Extracts of Chelidonium majus L. and Berberis thunbergii DC. toward Hematopoietic Cancer Cell Lines.

Isoquinoline alkaloids belong to the toxic secondary metabolites occurring in plants of many families. The high biological activity makes these compounds promising agents for use in medicine, particularly as anticancer drugs. The aim of our study was to evaluate the cytotoxicity and proapoptotic activity of sanguinarine, berberine, and extracts of Chelidonium majus L. and Berberis thunbergii DC. IC10, IC50, and IC90 doses were established toward hematopoietic cancer cell lines using trypan blue staining. Alterations in the expression of 18 apoptosis-related genes in cells exposed to IC10, IC50, and IC90 were evaluated using real-time PCR. Sanguinarine and Chelidonium majus L. extract exhibit significant cytotoxicity against all studied cell lines. Lower cytotoxic activity was demonstrated for berberine. Berberis thunbergii DC. extract had no influence on cell viability. Berberine, sanguinarine, and Chelidonium majus L. extract altered the expression of apoptosis-related genes in all tested cell lines, indicating the induction of apoptosis. The presented study confirmed the substantial cytotoxicity and proapoptotic activity of sanguinarine, berberine, and Chelidonium majus L. extract toward the studied hematopoietic cell lines, which indicates the utility of these substances in anticancer therapy.

Development of a Selective Adsorbing Material for Binding of Pyrrolizidine Alkaloids in Herbal Extracts, Based on Molecular Group Imprinting.

Pyrrolizidine alkaloids are secondary plant constituents that became a subject of public concern because of their hepatotoxic, pneumotoxic, genotoxic, and cytotoxic effects. Due to disregardful harvesting and/or contamination with pyrrolizidine alkaloid-containing plants, there is a high risk of ingesting these substances with plant extracts or natural products. The limit for the daily intake was set to 0.007 µg/kg body weight. If contained in an extract, cleanup methods may help to minimize the pyrrolizidine alkaloid concentration. For this purpose, a material for depleting pyrrolizidine alkaloids in herbal preparations was developed based on the approach of molecular imprinting using monocrotaline. Molecular imprinted polymers are substances with specific binding characteristics, depending on the template used for imprinting. By means of group imprinting, only one molecule is used for creating selective cavities for many molecular pyrrolizidine alkaloid variations. Design of Experiment was used for the development using a 25 screening plan resulting in 64 polymers (32 MIPs/32 NIPs). Rebinding trials revealed that the developed material can compete with common cation exchangers and is more suitable for depleting pyrrolizidine alkaloids than C18- material. Matrix trials using an extract from Chelidonium majus show that there is sufficient binding capacity for pyrrolizidine alkaloids (80%), but the material is lacking in selectivity towards pyrrolizidine alkaloids in the presence of other alkaloids with similar functional groups such as berberine, chelidonine, and coptisine. Beyond this interaction, the selectivity could be proven for other structurally different compounds on the example of chelidonic acid.

Protopine isolated from Nandina domestica induces apoptosis and autophagy in colon cancer cells by stabilizing p53.

The tumor suppressor p53 plays essential roles in cellular protection mechanisms against a variety of stress stimuli and its activation induces apoptosis or autophagy in certain cancer cells. Here, we identified protopine, an isoquinoline alkaloid isolated from Nandina domestica, as an activator of the p53 pathway from cell-based natural compound screening based on p53-responsive transcription. Protopine increased the p53-mediated transcriptional activity and promoted p53 phosphorylation at the Ser15 residue, resulting in stabilization of p53 protein. Moreover, protopine up-regulated the expression of p21WAF1/CIP1 and BAX, downstream genes of p53, and inhibited the proliferation of HCT116 colon cancer cells. Apoptosis was elicited by protopine as indicated by caspase-3/7 activation, poly ADP ribose polymerase cleavage, and increased population of Annexin V-FITC-positive cells. Furthermore, protopine induced the formation of microtubule-associated protein 1 light chain 3 (LC3) puncta and LC3-II turnover, typical biochemical markers of autophagy, in HCT116 cells. Our findings suggest that protopine exerts its antiproliferative activity by stimulating the p53 pathway and may have potential as a chemopreventive agent for human colon cancer.

Benzophenanthridine alkaloids from the roots of Thalictrum microgynum Lecoy.ex Oliv.

A new benzophenanthridine alkaloid, 2,3,9-trimethoxy-7,8-methylenedioxy-5-methylbenzo[c]-6(5H) phenanthridone (2) and a benzophenanthridine alkaloid first found from natural sources, 2,3-dimethoxy-7,8- methylenedioxy-5-methylbenzo[c]-6(5H)- phenanthridone (1) together with two known benzophenanthridine alkaloids, Dihydrosanguinarine (3) and Dihydrochelilutine (4) were isolated from the roots of Thalictrum microgynum Lecoy.ex Oliv. The structures of 1 and 2 were elucidated using various spectroscopic techniques including HRESIMS and 1 D and 2 D NMR. Antibacterial activity of these compounds were tested. Compound 1, 3 and 4 showed antibacterial activity against Staphylococcus aureus with MIC values of 50, 100, 25 µg/mL, respectively.

Hexahydrobenzophenanthridine alkaloids from Corydalis bungeana Turcz. and their anti-inflammatory activity.

As part of a bioprospecting program aimed at the discovery of anti-inflammatory agents from the Corydalis bungeana Turcz. (C. bungeana), five new hexahydrobenzophenanthridine alkaloids, corycaline A-E (1-5), along with four known alkaloids, were isolated from the whole plant of C. bungeana. Their structures including absolute configurations were elucidated on the basis of extensive spectroscopic analyses and electronic circular dichroism (ECD) calculations. The inhibitory activities of the nine compounds on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7 mouse macrophage cells were determined; all tested compounds except 2 and 7 exhibited significant inhibitory effects with IC50 values in the range of 1.00-2.79 µM.

Preparation and application of magnetic molecularly imprinted polymers for the isolation of chelerythrine from Macleaya cordata.

A novel type of magnetic molecularly imprinted polymer was prepared for the selective enrichment and isolation of chelerythrine from Macleaya cordata (Willd) R. Br. The magnetic molecularly imprinted polymers were prepared using functional Fe3 O4 @SiO2 as a magnetic support, chelerythrine as template, methacrylic acid as functional monomer, and ethylene glycol dimethacrylate as cross-linker. Density functional theory at the B3LYP/6-31G (d, p) level with Gaussian 09 software was applied to calculate the interaction energies of chelerythrine, methacrylic acid and the complexes formed from chelerythrine and methacrylic acid in different ratios. The structural features and morphology of the synthesized polymers were characterized by using Fourier transform infrared spectroscopy, X-ray diffraction, transmission electron microscopy, and vibration sample magnetometry. Adsorption experiments revealed that the magnetic molecularly imprinted polymers possessed rapid kinetics, high selectivity, and a higher binding capacity (7.96 mg/g) to chelerythrine than magnetic molecularly non-imprinted polymers (2.36 mg/g). The adsorption process was in good agreement with the Langmuir adsorption isotherm and pseudo-second-order kinetics models. Furthermore, the magnetic molecularly imprinted polymers were successfully employed as adsorbents for the extraction and enrichment of chelerythrine from Macleaya cordata (Willd) R. Br. The results indicated that the magnetic molecularly imprinted polymers were suitable for the selective adsorption of chelerythrine from complex samples such as natural medical plants.

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.

Enrichment of chelidonine from Chelidonium majus L. using macroporous resin and its antifungal activity.

Chelidonium majus L. (greater celandine) has been used as an herbal medicine for several centuries. This study investigated an efficient method to purify chelidonine from the extract of C. majus L. using macroporous adsorption resins and evaluated the antifungal activity of chelidonine against Botryosphaeria dothidea as a model strain. Static adsorption and desorption tests revealed that D101 was the optimal resin for chelidonine purification. Pseudo-second-order kinetics model and Freundlich equation model were the most suitable for evaluating the endothermic and spontaneous adsorption processes of chelidonine on D101. Dynamic adsorption and desorption tests on D101 columns showed that the concentration of chelidonine increased 14.16-fold, from 2.67% to 37.81%, with the recovery yield of 80.77%. The antifungal activity of enriched chelidonine products was studied with B. dothidea. The results showed that the EC50 of crude extracts, enriched chelidonine products, and chelidonine standard against B. dothidea were 3.24mg/mL, 0.43mg/mL, and 0.77mg/mL, respectively. The result of antifungal activity test showed that chelidonine had the potential to be a useful antifungal agent. Moreover, the enrichment method of chelidonine was highly efficient, low cost, and harmless to the environment for industrial applications.

Evaluation of the Anticancer Activities of the Plant Alkaloids Sanguinarine and Chelerythrine in Human Breast Adenocarcinoma Cells.

BACKGROUND: Breast cancer is associated with a high mortality rate around the world due to its aggressiveness and high resistance to conventional therapies. Sanguinarine (SAN) and Chelerythrine (CHE) are plant alkaloids extracted from Sanguinaria canadensis and Macleaya cordata, which have been studied for their bioactivities. OBJECTIVE: To determine the anticancer activities of Sanguinarine (SAN) and Chelerythrine (CHE) plant alkaloids. METHOD: The MTT assay, the alkaline comet assay and cell cycle analyses by flow cytometry were performed. RESULTS: It was observed that SAN was cytotoxic to human breast adenocarcinoma cells (MCF-7) at concentrations of 7.5 µM (24 and 48 hours), effectively reducing cell viability from the concentration of 10 µM for 24 hours and 7.5 µM for 48 hours, by the MTT test. CHE, in turn, was cytotoxic at concentrations of 10 and 20 µM (48 hours), but did not compromise the cellular viability. The comet assay indicated that SAN was genotoxic to the MCF-7 cells, with a significant increment of damage at 10 µM, while none of the tested concentrations of CHE showed a genotoxic effect. The flow cytometry analysis indicated that no cell cycle arrest was caused by both alkaloids, but SAN 10 µM induced a sub-G1 cell population. CONCLUSION: The results of cytotoxicity, genotoxicity and cell cycle monitoring that are presented in this paper have suggested that SAN has more of a chemotherapeutic activity, as well as having the potential for the development of new therapies for breast cancer, when compared to CHE.

The gene expression profiles in response to 102 traditional Chinese medicine (TCM) components: a general template for research on TCMs.

Traditional Chinese medicines (TCMs) have important therapeutic value in long-term clinical practice. However, because TCMs contain diverse ingredients and have complex effects on the human body, the molecular mechanisms of TCMs are poorly understood. In this work, we determined the gene expression profiles of cells in response to TCM components to investigate TCM activities at the molecular and cellular levels. MCF7 cells were separately treated with 102 different molecules from TCMs, and their gene expression profiles were compared with the Connectivity Map (CMAP). To demonstrate the reliability and utility of our approach, we used nitidine chloride (NC) from the root of Zanthoxylum nitidum, a topoisomerase I/II inhibitor and α-adrenoreceptor antagonist, as an example to study the molecular function of TCMs using CMAP data as references. We successfully applied this approach to the four ingredients in Danshen and analyzed the synergistic mechanism of TCM components. The results demonstrate that our newly generated TCM data and related methods are valuable in the analysis and discovery of the molecular actions of TCM components. This is the first work to establish gene expression profiles for the study of TCM components and serves as a template for general TCM research.

Sanguinarine Induces Apoptosis of Human Oral Squamous Cell Carcinoma KB Cells via Inactivation of the PI3K/Akt Signaling Pathway.

Preclinical Research Sanguinarine, an alkaloid isolated from the root of Sanguinaria canadensis and other plants of the Papaveraceae family, selectively induces apoptotic cell death in a variety of human cancer cells, but its mechanism of action requires further elaboration. The present study investigated the pro-apoptotic effects of sanguinarine in human oral squamous cell carcinoma KB cells. Sanguinarine treatment increased DR5/TRAILR2 (death receptor 5/TRAIL receptor 2) expression and enhanced the activation of caspase-8 and cleavage of its substrate, Bid. Sanguinarine also induced the mitochondrial translocation of pro-apoptotic Bax, mitochondrial dysfunction, cytochrome c release to the cytosol, and activation of caspase-9 and -3. However, a pan-caspase inhibitor, z-VAD-fmk, reversed the growth inhibition and apoptosis induced by sanguinarine. Sanguinarine also suppressed the phosphorylation of phosphoinositide 3-kinase (PI3K) and Akt in KB cells, while co-treatment of cells with sanguinarine and a PI3K inhibitor revealed synergistic apoptotic effects. However, pharmacological inhibition of AMP-activated protein kinase and mitogen-activated protein kinases did not reduce or enhance sanguinarine-induced growth inhibition and apoptosis. Collectively, these findings indicate that the pro-apoptotic effects of sanguinarine in KB cells may be regulated by a caspase-dependent cascade via activation of both intrinsic and extrinsic signaling pathways and inactivation of PI3K/Akt signaling. Drug Dev Res 77 : 227-240, 2016. © 2016 Wiley Periodicals, Inc.

Alkaloid profiles and acetylcholinesterase inhibitory activities of Fumaria species from Bulgaria.

GC-MS analysis of alkaloid profiles of five Fumaria species, naturally grown in Bulgaria (F. officinalis, F. thuretii, F. kralikii, F. rostellata and F. schrammii) and analysis of acetylcholinesterase inhibitory activity of alkaloid extracts were performed. Fourteen isoquinoline alkaloids were identified, with the principle ones being protopine, cryptopine, sinactine, parfumine, fumariline, fumarophycine, and fumaritine. Protopine contents, defined by HPLC analysis varied between 210.6 ± 8.8 µg/g DW (F. schrammii) and 334.5 ± 7.1 µg/g DW. (F. rostellata). While all of the investigated alkaloid extracts significantly inhibited acetylcholinesterase activity, the F. kralikii demonstrated the highest level of inhibition (IC(50) 0.13 ± 0.01 mg extract/mL).

A new benzophenanthridine alkaloid and other bioactive constituents from the stem bark of Zanthoxylum heitzii.

Heitziquinone (7), a new benzophenanthridine alkaloid, together with five known compounds; isoarnottianamide (5), rhoifoline B (6), isobauerenol (8), 6-hydroxypellitorine (9) and sylvamide (10), were isolated as minor compounds from the hexane extract of stem bark from Zanthoxylum heitzii. Four previously reported compounds (1-4) were found, as well. Compounds 5 and 7 were both found to exist as 4:1 mixtures of two atropisomers. The structures were elucidated by 1D and 2D NMR spectroscopy and by mass spectrometry. Compounds 5-10 were identified for the first time in this species, and they are all rare natural compounds. Pellitorine (4), one of the main compounds from the hexane bark extract, was found to be responsible for the brine shrimp larvae toxicity (LC50 37 µM, 8 µg/ml) of the crude extract (LC50 24 µg/ml). Low cytotoxicity against a macrophage cell line was observed.

Characterization of l-Digitoxosyl-phenanthroviridin from Streptomyces venezuelae ISP5230.

The jadomycin-derived compound l-digitoxosyl-phenanthroviridin was isolated from fermentations of Streptomyces venezuelae ISP5230 grown in nutrient-deficient media with l-lysine as the sole nitrogen source. Structural elucidation was accomplished using a combination of high-resolution MS, LC-MS/MS, and 1D- and 2D-NMR. The compound was evaluated against the National Cancer Institute (NCI) 60 human tumor cell line screen in both the one-dose and five-dose screens, and cytotoxicity was compared to a small library of jadomycin analogues to probe the structure-activity relationship.

Antibacterial activities of the methanol extracts, fractions and compounds from Fagara tessmannii.

ETHNOPHARMACOLOGICAL RELEVANCE: Fagara tessmannii is a shrub of the African rainforests used to treat bacterial infections, cancers, swellings and inflammation. In the present study, the methanol extract from the leaves (FTL), bark (FTB), and roots (FTR) of this plant as well as fractions (FTR1-5) and compounds isolated from FTR namely ß-sitosterol-3-O-ß-d-glucopyranoside (1), nitidine chloride (2) and buesgenine (3), were tested for their antimicrobial activities against a panel of Gram-negative bacteria including multidrug resistant (MDR) phenotypes. MATERIALS AND METHODS: The broth microdilution method was used to determine the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of the above samples; Column chromatography was used for the fractionation and purification of the roots extract whilst the chemical structures of compounds were determined using spectroscopic techniques. RESULTS: Results of the MIC determinations indicated that the crude extracts from the roots as well as fraction FTRa4 were active on all the 26 tested bacterial strains. MIC values below 100µg/mL were obtained with roots, leaves and bark extract respectively against 30.8%, 15.4% and 11.5% tested bacteria. The lowest MIC value below of 8µg/mL was obtained with extract from the roots against Escherichia coli MC100 strain. The lowest MIC value of 4µg/mL was also obtained with compound 3 against E. coli AG102 and Klebsiella pneumoniae ATCC11296 CONCLUSIONS: The present study demonstrates that F. tessmannii is a potential source of antimicrobial drugs to fight against MDR bacteria. Benzophenanthrine alkaloids 2 and 3 are the main antibacterial consituents of the roots of the plant.

A new cytotoxic benzophenanthridine isoquinoline alkaloid from the fruits of Macleaya cordata.

A new cytotoxic benzophenanthridine isoquinoline alkaloid, named cordatine (1), together with one known alkaloid 8-methoxydihydrochelerythrine (2), was isolated from the fruits of Macleaya cordata. The structure of the new compound was elucidated by spectroscopic methods including 1D and 2D NMR, HR-ESI-MS. Both compounds indicated significant cytotoxicity against MCF-7 and SF-268 cell lines.

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.

Leptopyrine, new alkaloid from Leptopyrum fumarioides L. (Ranunculaceae).

A new type of isoquinoline alkaloid leptopyrine was isolated from the aerial parts of Leptopyrum fumarioides L. (Ranunculaceae) of Mongolian origin. The known alkaloids protopine and thalifoline were isolated for the first time from this the species. All structures were established by physical and spectral analyses.