Variability of Isoquinoline Alkaloid Profiles and Anticholinesterase Activities with Binding-Mode Predictions of Glaucium flavum Population.

In this study, phytochemical and biological activity studies supported by docking were carried out on a species of the genus Glaucium, a repository of isoquinoline alkaloids. The GC-MS (Gas Chromatography-Mass Spectrometry) method is used to characterize the isoquinoline alkaloids of Glaucium flavum Crantz. (Papaveraceae). G. flavum was collected from seven different regions of Türkiye (Antalya, Urla-Izmir, Mordogan-Izmir, Mugla, Assos-Canakkale, Karabiga-Canakkale, Giresun) and totally 17 compounds were detected by GC-MS. Glaucine was found to be the major constituent in the sample collected from Mugla, whereas isocorydine was recorded to be the principal alkaloid in other samples. Further fractionation studies on G. flavum collected from Antalya province in Southwestern Türkiye, yielded five major alkaloids (isocorydine 1, dihydrosanguinarine 2, glaucine 3, dehydroglaucine 4, protopine 5) which were characterized by spectroscopic methods. Anticholinesterase activities of the extracts and isolated alkaloids were also tested by in vitro Ellman method. The isolated compounds were also analyzed by a molecular docking technique to determine the binding orientations in the gorge of the active site of acetylcholinesterase (AChE) and a homology model of butyrylcholinesterase (BuChE). This is the first comparative investigation of the phytochemical composition and biodiversity of Glaucium flavum species growing in Türkiye.

Determination of Some Isoquinoline Alkaloids in Extracts Obtained from Selected Plants of the Ranunculaceae, Papaveraceae and Fumarioideae Families by Liquid Chromatography and In Vitro and In Vivo Investigations of Their Cytotoxic Activity.

Alkaloids are heterocyclic bases with widespread occurrence in nature. Plants are rich and easily accessible sources of them. Most isoquinoline alkaloids have cytotoxic activity for different types of cancer, including malignant melanoma, the most aggressive type of skin cancer. The morbidity of melanoma has increased worldwide every year. For that reason, developing new candidates for anti-melanoma drugs is highly needed. The aim of this study was to investigate the alkaloid compositions of plant extracts obtained from Macleaya cordata root, stem and leaves, Pseudofumaria lutea root and herb, Lamprocapnos spectabilis root and herb, Fumaria officinalis whole plant, Thalictrum foetidum root and herb, and Meconopsis cambrica root and herb by HPLC-DAD and LC-MS/MS. For determination of cytotoxic properties, human malignant melanoma cell line A375, human Caucasian malignant melanoma cell line G-361, and human malignant melanoma cell line SK-MEL-3 were exposed in vitro to the tested plant extracts. Based on the in vitro experiments, Lamprocapnos spectabilis herb extract was selected for further, in vivo research. The toxicity of the extract obtained from Lamprocapnos spectabilis herb was tested using an animal zebrafish model in the fish embryo toxicity test (FET) for determination of the LC50 value and non-toxic doses. Determination of the influence of the investigated extract on the number of cancer cells in a living organism was performed using a zebrafish xenograft model. Determination of the contents of selected alkaloids in different plant extracts was performed using high performance liquid chromatography (HPLC) in a reverse-phase system (RP) on a Polar RP column with a mobile phase containing acetonitrile, water and ionic liquid. The presence of these alkaloids in plant extracts was confirmed by LC-MS/MS. Preliminary cytotoxic activity of all prepared plant extracts and selected alkaloid standards was examined using human skin cancer cell lines A375, G-361, and SK-MEL-3. The cytotoxicity of the investigated extract was determined in vitro by cell viability assays (MTT). For in vivo determination of investigated extract cytotoxicity, a Danio rerio larvae xenograft model was used. All investigated plant extracts in in vitro experiments exhibited high cytotoxic activity against the tested cancer cell lines. The results obtained using the Danio rerio larvae xenograft model confirmed the anticancer activity of the extract obtained from Lamprocapnos spectabilis herb. The conducted research provides a basis for future investigations of these plant extracts for potential use in the treatment of malignant melanoma.

Isoquinoline alkaloids isolated from Glaucium corniculatum var. corniculatum and Glaucium grandiflorum subsp. refractum var. torquatum with bioactivity studies.

CONTEXT: The genus Glaucium Mill., one of the important Papaveraceae family plants, is rich in isoquinoline alkaloids and distributed worldwide. OBJECTIVE: Isolation and identification of bioactive alkaloids from Glaucium grandiflorum Boiss. & Huet. subsp. refractum (Nabelek) Mory var. torquatum (Cullen) Mory and G. corniculatum (L.) Rudolph var. corniculatum (Aslan 2012), and investigation of their antioxidant and anticholinesterase activities. MATERIALS AND METHODS: The aerial parts of each plant were dried, powdered, and percolated with methanol, then each extract was fractionated between 50% aqueous acetic acid and petroleum. Their aqueous acidic layer was adjusted to pH 7-8 with NH4OH and extracted with chloroform, the extract was subjected to CC separation and isolation. Structures of the isolated alkaloids were elucidated by 1D and 2D-NMR and mass spectral analyses. The alkaloid extracts and their pure alkaloids were tested for anti-cholinesterase (AChE and BuChE) and antioxidant (ABTS, CUPRAC, ß-carotene linoleic acid tests) activities in vitro. RESULTS: Methanol extracts of Glaucium grandiflorum subsp. refractum var. torquatum and G. corniculatum var. corniculatum afforded a novel compound glauciumoline and seven known isoquinoline alkaloids three of which have an aporphine-type and the other five have a protopine-type skeleton. Among them, trans-protopinium (7) and cis-protopinium (8) were isolated from a Glaucium species for the first time. Tertiary amine extracts (TAEs) of both plants showed very strong acetylcholinesterase inhibitory activity. The TAE of the plants also showed strong antioxidant activity while the isolated alkaloids showed no meaningful activity in the anticholinesterase and antioxidant tests. DISCUSSION AND CONCLUSIONS: Glaucium species are considered promising therapeutic agents in the treatment of Alzheimer's disease.

Sex differences in the pharmacokinetics and tissue residues of Macleaya cordata extracts in rats.

Macleaya cordata extracts (MCE) are listed as feed additives in animal production by the European Food Authority. The core components of MCE are mainly sanguinarine (SA) and chelerythrine (CHE). This study aims to investigate sex differences in the pharmacokinetics and tissue residues of MCE in rats.Male and female rates were intragastrically administered MCE (1.25 mg·kg-1 body weight and 12.5 mg·kg-1 body weight dose for 28 days). SA and CHE concentrations were determined using high-performance liquid chromatography/tandem mass spectrometry.The peak plasma concentration (Cmax) and area under the curve (AUC) of both CHE and SA were higher in female than in male rats (12.5 mg·kg-1 body weight group), whereas their half-life (T1/2) and apparent volume of distribution (Vd) was lower (p < 0.05). Tissue rfesidue analysis indicated that SA and CHE were more distributed in male than in female rats and were highly distributed in the caecum and liver. SA and CHE were completely eliminated from the liver, kidney, lung, heart, spleen, leg muscle, and caecum after 120 h, indicating they did not accumulate in rats for a long time.Overall, we found that the pharmacokinetics and tissue residues of SA and CHE of male and female rats showed sex differences.

Isoquinoline Alkaloid Contents in Macleaya cordata Extracts and Their Acetylcholinesterase and Butyrylcholinesterase Inhibition.

An important strategy for treating neurodegenerative disorders is to maintain the levels of acetylcholine in the synaptic cleft by blocking the cholinesterases. Searching for new effective compounds with inhibited acetylcholinesterase and butyrylcholinesterase activity is one of the most significant challenges of the modern scientific research. The aim of this study was the optimization of the condition for cholinesterase activity determination by high-performance liquid chromatography coupled with diode array detector (HPLC-DAD) in terms of concentrations of enzymatic reaction mixture components, temperature of incubation, and incubation time. In vitro investigation of acetylcholinesterase and butyrylcholinesterase activity inhibition by some isoquinoline alkaloids and extracts obtained from the aerial part and roots of Macleaya cordata collected in May, July, and September. Acetylcholinesterase and butyrylcholinesterase activity inhibition of the extracts obtained from the plant had not been tested previously. The application of the HPLC method allowed eliminating absorption of interfering components, for example, alkaloids such as sanguinarine and berberine. The HPLC method was successfully applied for the evaluation of the acetylcholinesterase inhibitory activity in samples such as plant extracts, especially those containing colored components adsorbing at the same wavelength as the adsorption wavelength of 5-thio-2-nitro-benzoic acid, which is the product of the reaction between thiocholine (product of the hydrolysis of acetyl/butyrylthiocholine reaction) with Ellman's reagent. Moreover, liquid chromatography coupled with a triple quadrupole mass spectrometer (LC-QqQ-ESI-MS/MS) analysis allowed evaluating the identification of relevant bioactive compounds in the obtained plant extracts. The investigated alkaloids, especially sanguinarine and chelerythrine, and all the Macleaya cordata extracts, especially the extract obtained from the aerial part collected in May, exhibited very high cholinesterase activity inhibition. HPLC-DAD was also applied for the kinetics study of the most active alkaloids sanguinarine and chelerythrine. Our investigations demonstrated that these plant extracts can be recommended for further in vivo experiments to confirm their cholinesterase inhibition activity.

An Integrated-Omics/Chemistry Approach Unravels Enzymatic and Spontaneous Steps to Form Flavoalkaloidal Nudicaulin Pigments in Flowers of Papaver nudicaule L.

Flower colour is an important trait for plants to attract pollinators and ensure their reproductive success. Among yellow flower pigments, the nudicaulins in Papaver nudicaule L. (Iceland poppy) are unique due to their rarity and unparalleled flavoalkaloid structure. Nudicaulins are derived from pelargonidin glycoside and indole, products of the flavonoid and indole/tryptophan biosynthetic pathway, respectively. To gain insight into the molecular and chemical basis of nudicaulin biosynthesis, we combined transcriptome, differential gel electrophoresis (DIGE)-based proteome, and ultra-performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS)-based metabolome data of P. nudicaule petals with chemical investigations. We identified candidate genes and proteins for all biosynthetic steps as well as some key metabolites across five stages of petal development. Candidate genes of amino acid biosynthesis showed a relatively stable expression throughout petal development, whereas most candidate genes of flavonoid biosynthesis showed increasing expression during development followed by downregulation in the final stage. Notably, gene candidates of indole-3-glycerol-phosphate lyase (IGL), sharing characteristic sequence motifs with known plant IGL genes, were co-expressed with flavonoid biosynthesis genes, and are probably providing free indole. The fusion of indole with pelargonidin glycosides was retraced synthetically and promoted by high precursor concentrations, an excess of indole, and a specific glycosylation pattern of pelargonidin. Thus, nudicaulin biosynthesis combines the enzymatic steps of two different pathways with a spontaneous fusion of indole and pelargonidin glycoside under precisely tuned reaction conditions.

Screening Papaveraceae as Novel Antibiofilm Natural-Based Agents.

The antimicrobial properties of herbs from Papaveraceae have been used in medicine for centuries. Nevertheless, mutual relationships between the individual bioactive substances contained in these plants remain poorly elucidated. In this work, phytochemical composition of extracts from the aerial and underground parts of five Papaveraceae species (Chelidonium majus L., Corydalis cava (L.) Schweigg. and Körte, C. cheilanthifolia Hemsl., C. pumila (Host) Rchb., and Fumaria vaillantii Loisel.) were examined using LC-ESI-MS/MS with a triple quadrupole analyzer. Large differences in the quality and quantity of all analyzed compounds were observed between species of different genera and also within one genus. Two groups of metabolites predominated in the phytochemical profiles. These were isoquinoline alkaloids and, in smaller amounts, non-phenolic carboxylic acids and phenolic compounds. In aerial and underground parts, 22 and 20 compounds were detected, respectively. These included: seven isoquinoline alkaloids: protopine, allocryptopine, coptisine, berberine, chelidonine, sanguinarine, and chelerythrine; five of their derivatives as well as non-alkaloids: malic acid, trans-aconitic acid, quinic acid, salicylic acid, trans-caffeic acid, p-coumaric acid, chlorogenic acid, quercetin, and kaempferol; and vanillin. The aerial parts were much richer in phenolic compounds regardless of the plant species. Characterized extracts were studied for their antimicrobial potential against planktonic and biofilm-producing cells of S. aureus, P. aeruginosa, and C. albicans. The impact of the extracts on cellular metabolic activity and biofilm biomass production was evaluated. Moreover, the antimicrobial activity of the extracts introduced to the polymeric carrier made of bacterial cellulose was assessed. Extracts of C. cheilanthifolia were found to be the most effective against all tested human pathogens. Multiple regression tests indicated a high antimicrobial impact of quercetin in extracts of aerial parts against planktonic cells of S. aureus, P. aeruginosa, and C. albicans, and no direct correlation between the composition of other bioactive substances and the results of antimicrobial activity were found. Conclusively, further investigations are required to identify the relations between recognized and unrecognized compounds within extracts and their biological properties.

Synthesis and characterization of core-shell magnetic molecularly imprinted polymers for selective extraction of allocryptopine from the wastewater of Macleaya cordata (Willd) R. Br.

A novel type of magnetic molecularly imprinted polymers (MMIP) as the solid-phase extraction sorbent was prepared, which can extract effectively the allocryptopine from the waster of Macleaya cordata (Willd) R. Br. In this study, MMIP was synthesized by using Fe3 O4 @SiO2 , 4-vinyl-pyridine, ethylene glycol dimethacrylate, and allocryptopine, and these ingredients worked as magnetic core, functional monomer, cross-linker, and template, respectively. Concluded by the calculation of Gaussian 09 software, different ratio models of 4-vinyl-pyridine and allocryptopine were simulated, and the optimal ratio was 1:5 and the energy was -2205.34 kJ/mol. Transmission electron microscopy, vibration sample magnetometry, X-ray diffraction, Fourier transform infrared spectroscopy, and thermogravimetric analysis were used to determine the morphology and structure of MMIP. Furthermore, the results of adsorption experiments indicated that MMIP had high selectivity, excellent recyclability, and good adsorption performance (9.86 mg/g, 298 K). The adsorption process was consistent with the Langmuir adsorption isotherm (R2 > 0.98, 298 K) and pseudo-second-order kinetics model (R2 > 0.99, 298 K). After six times adsorption-desorption experiments, the adsorption amount of MMIP only reduced to 8.5%. In the experiments of selective adsorption, MMIP has better adsorption properties for allocryptopine (ALL, C21 H23 NO5 ) than those having the same functional group. The limit of detection (LOD) was 0.4 µg/mL. The relative standard deviation ranged from 0.09% to 0.72%. The recovery of allocryptopine in samples ranged from 93.60% to 106.19%. In addition, the synthesized complex had a certain adsorption effect on allocryptopine separating from the wastewater of Macleaya cordata (Willd) R. Br.

Identification of drought resistant miRNA in Macleaya cordata by high-throughput sequencing.

Drought is one of the most serious factors affecting crop yields in the world. Macleaya cordata (Willd.) is a draught-tolerant medicinal plant that has been proposed as a pioneer crop to be cultivated in arid areas. However, the exact molecular mechanisms through which M. cordata responds to draught stress remain elusive. In recent years, microRNA (miRNAs) in plants have been associated with stress response. Based on these findings, the current study aimed to shed light on the potential regulatory roles of miRNAs in the draught tolerance of M. cordata by employing high-throughput RNA sequencing and degradation sequencing. Six M. cordata plants were randomly divided into two equal experiment groups, including one draught group and one control group. High-throughput sequencing of the M. cordata samples led to the identification of 895 miRNAs, of which 18 showed significantly different expression levels between the two groups. PsRobot analysis and degradation sequencing predicted the differential miRNAs to target 59 and 36 genes, respectively. Functional analysis showed that 38 of the predicted genes could be implicated in the modulation of stress response. Four miRNAs and eight target genes were selected for quantitative real-time polymerase chain reaction (qRT-PCR) validation. The expression trend of each miRNA analyzed by qRT-PCR was consistent with that determined by sequencing, and was negatively correlated with those of its target genes. The results of our current study supported the involvement of miRNAs in the draught tolerance of M. cordata and could pave the way for further investigation into the related regulatory mechanisms.

Systematic characterization of alkaloids in Eomecon chionantha Hance using ultrahigh-performance liquid chromatography-tandem quadrupole Exactive Orbitrap mass spectrometry with a four-step screening strategy.

RATIONALE: Eomecon chionantha Hance (ECH), a traditional folk herb, is commonly used to treat traumatic injuries based on its analgesic and anti-inflammatory properties. Previous studies have reported that alkaloids are the major bioactive components in ECH. Therefore, identification of alkaloids from ECH contributes to the discovery of its potential active ingredients and quality control in clinic treatments. METHODS: A four-step screening strategy was performed as follows. (1) Extracting the accurate masses of ions related to different molecules. (2) Screening different types of compounds using their molecular cations, protonated molecules, diagnostic product ions and fragmentation pathways. (3) Comparing the characteristic product ion formulae to obtain the type and number of substituents. (4) Using the biosynthetic pathways of isoquinoline alkaloids to determine the concentration of alkaloids. RESULTS: Ultrahigh-performance liquid chromatography-tandem quadrupole Exactive Orbitrap mass spectrometry (UHPLC/Q-Exactive Orbitrap MS) analysis combined with the four-step screening strategy was used to profile the alkaloids in ECH. The structures of 95 alkaloids in ECH were unambiguously identified or reasonably assigned, of which 76 were reported in ECH for the first time. Six types of benzylisoquinoline alkaloids were identified in ECH: six benzyltetrahydroisoquinolines, nine protopines, five N-methyltetrahydroprotoberberines, six protoberberines, eight benzophenanthridines and sixty-one dihydrobenzophenanthridines. CONCLUSIONS: This comprehensive study identified the alkaloids in ECH, thus providing a practical reference for further research. The UHPLC/Q-Exactive Orbitrap MS method, combined with the four-step screening strategy, which was developed and successfully applied to identify the alkaloids in ECH, may also be applicable for the efficient screening of other herbal medicines.

Chemical constituents from Macleaya cordata (Willd) R. Br. and their phenotypic functions against a Parkinson's disease patient-derived cell line.

Cytological profiling (CP) assay against a human olfactory neuroshpere-derived (hONS) cell line using a library of traditional Chinese medicinal plant extracts gave indications that the ethanolic extract of Macleaya cordata (Willd) R. Br. elicited strong perturbations to various cellular components. Further chemical investigation of this extract resulted in the isolation of two new benzo[c]phenanthridine alkaloids, (6R)-10-methoxybocconoline (1) and 6-(1-hydroxyethyl)-10-methoxy-5,6-dihydrochelerythrine (2). Their planar structures were elucidated by extensive 1D and 2D NMR studies, together with MS data. The absolute configuration for position C-6 of 1 and relative configurations for position C-6 and C-1' of 2 were assigned by density functional theory (DFT) calculations of ECD and NMR data, respectively. Also isolated were fourteen known metabolites, including ten alkaloids (3-12) and four coumaroyl-containing compounds (13-16). Cytological profiling of the isolates against Parkinson's Disease (PD) patient-derived olfactory cells revealed bocconoline (3) and 6-(1-hydroxyethyl)-5,6-dihydrochelerythrine (4) significantly perturbated the features of cellular organelles including early endosomes, mitochondria and autophagosomes. Given that hONS cells from PD patients model some functional aspects of the disease, the results suggested that these phenotypic profiles may have a role in the mechanisms underlying PD and signified the efficacy of CP in finding potential chemical tools to study the biological pathways in PD.

Online high-pH reversed-phase liquid chromatography × low-pH reversed-phase liquid chromatography tandem electrospray ionization mass spectrometry combined with pulse elution gradient in the first dimension for the analysis of alkaloids in Macleaya cordata (willd.) R. Br.

An online high-pH reversed-phase liquid chromatography× low-pH reversed-phase liquid chromatography tandem electrospray ionization mass spectrometry combined with pulse elution gradient in the first dimension was constructed to separate and identify alkaloids from Macleaya cordata (willd.) R. Br. The modulation was performed by using a dual second dimensional columns interface combined with a make-up dilution pump, which is responsible for dilution and neutralization of the first dimensional effluent, and the dual second dimensional columns integrated the trapping and the separation function to reduce the second dimension system dead volume. Taking advantage of the dissociable characteristics of alkaloids, mobile phases with different pH values were applied in the first dimension (pH 9.0) and the second dimension (pH 2.6) to improve the orthogonality of two-dimension separation. Besides, the pulse elution gradient in first dimension and second dimensional gradient were carefully optimized and much better separation was achieved compared to the separation with the traditional two-dimensional liquid chromatography approach. Finally, mass measurement was performed for alkaloids in M. cordata (willd.) R. Br. by coupling proposed two-dimensional liquid chromatography system with triple quadrupole mass spectrometry, and 39 alkaloids were successfully identified by comparing the obtained result with the former reported results.

Isolation and purification of alkaloids from the fruits of Macleaya cordata by ionic-liquid-modified high-speed counter-current chromatography.

Macleaya cordata (Willd) R. Br. is a medicinal plant. The most important bioactive compounds of M. cordata are alkaloids that have many biological activities including antifungal, anti-inflammatory, and antitumor. In this study, an ionic-liquid-modified high-speed counter-current chromatography method was established to obtain alkaloids from the fruits of M. cordata. The conditions of ionic-liquid-modified high-speed counter-current chromatography, including solvent systems, the content of ionic liquid (1-butyl-3-methylimidazolium tetrafluoroborate [C4 mim][BF4 ]), and the posttreatment of the ionic liquid, were investigated. Five alkaloids protopine, allocryptopine, sanguinarine, 8-O-demethylchelerythrine, and chelerythrine were separated from the extract of the fruits using a high speed counter-current chromatography with two-phase solvent system composed of dichloromethane/methanol/0.3 mol/L hydrochloric acid aqueous solution/[C4 mim][BF4 ] (4:2:2:0.015, v/v). Their purities were 96.33, 95.56, 97.94, 96.22, and 97.90%, respectively. The results indicated that a small amount of ionic liquids as modifier of the two-phase solvent system could shorten the separation time and improve the separation efficiency of the alkaloids from the fruits. The ionic-liquid-modified high-speed counter-current chromatography would provide a feasible way for highly effective separation of alkaloids from natural products.

Effects of isoquinoline alkaloids from Macleaya cordata on physiological, immunological and inflammatory parameters in healthy beagles: Alkaloids in dog nutrition.

The aim of the study was to investigate nutritional, physiological and immunological effects of a plant-derived blend of isoquinoline alkaloids (Sangrovit® Extra) in healthy dogs. Two groups of healthy, adult beagles (N = 10) were tested in a cross-over experiment, lasting two consecutive three-week periods. The experimental group received 1.2 g additive/kg feed, according to the recommendation of 10-20 mg/kg live weight per day. The control group received the same feed without additive. Complete blood count, immunological parameters and amino acid concentrations in serum were assessed. Faeces were analysed for short-chain fatty acids, lactate and ammonium; moreover, their quantity and consistency were determined. Neither feed intake, total apparent nutrient digestibility (crude protein and fat, organic matter, sodium, potassium) were affected by intake of the product. Lymphocyte and monocyte counts were slightly increased in both groups. Elevation was not treatment dependant. IgA, IgG, haptoglobin in serum and flow cytometric phenotyping of peripheral lymphocytes were not affected by alkaloids supplementation. Numerically greater methionine concentrations in blood serum occurred in the experimental group (p = 0.182). Quantity and consistency of faeces and ammonium concentration in faeces were not affected by the additive. Faecal concentrations of short-chain organic acids differed between groups (acetic acid, % of total SCFA: control group 52.3 ± 5.2 vs. experimental group 57.1 ± 4.5, p = 0.042), lactate concentrations (d-, l- and total) did not. Due to the shift of SCFA proportions in faeces, an effect of isoquinoline alkaloids (IQs) on the metabolic activity of intestinal microbiota is probable. In conclusion, the addition of IQs in the given dose was well tolerated and did not have adverse effects in healthy dogs.

Sanguinarine triggers intrinsic apoptosis to suppress colorectal cancer growth through disassociation between STRAP and MELK.

BACKGROUND: Previous studies showed sanguinarine induced apoptosis in CRC cells but did not define the underlying mechanisms. The purpose of this work was to determine the in vivo and in vitro effects of sanguinarine on CRC tumors and to elucidate the mechanism in regulating the intrinsic apoptosis. METHODS: Cell viability of CRC cell lines treated with sanguinarine was measured by MTT assay. Apoptotic cells stained with Annexin V and 7-AAD were detected by flow cytometry. Mitochondrial membrane potential and reactive oxygen species (ROS) were analyzed by JC-1 and DCFH-DA staining, respectively. The in vitro kinase activity of MELK was analyzed by using HTRF® KinEASE™-STK kit. The expression of proteins were determined using Western blotting and immunohistochemistry. Co-immunoprecipitation and immunofluorecence were used to study the interaction between STRAP and MELK. The anti-neoplastic effect of sanguinarine was observed in vivo in an orthotopic CRC model. RESULTS: Sanguinarine decreased the tumor size in a dose-dependent manner in orthotopical colorectal carcinomas through intrinsic apoptosis pathway in BALB/c-nu mice. It significantly increased cleavage of caspase 3 and PARP in implanted colorectal tissues. Sanguinarine increased mitochondrial ROS and triggered mitochondrial outer membrane permeabilization in multiple colorectal cancer (CRC) cell lines. NAC pretreatment lowered ROS level and downregulated apoptosis induced by sanguinarine. The intrinsic apoptosis induced by sanguinarine was Bax-dependent. The elevated expression and association between serine-threonine kinase receptor-associated protein (STRAP) and maternal embryonic leucine zipper kinase (MELK) were observed in Bax positive cells but not in Bax negative cells. Sanguinarine dephosphorylated STRAP and MELK and disrupted the association between them in HCT116 and SW480 cells. The expression and association between STRAP and MELK were also attenuated by sanguinarine in the tumor tissues. Importantly, we found that STRAP and MELK were overexpressed and highly phosphorylated in colorectal adenocarcinomas and their expression were significantly correlated with tumor stages. Furthermore, the expression of MELK, but not STRAP, was associated with lymph node metastasis. CONCLUSIONS: Sanguinarine dephosphorelates STRAP and MELK and disassociates the interaction between them to trigger intrinsic apoptosis. Overexpression of STRAP and MELK may be markers of CRC and their disassociation may be a determinant of therapeutic efficacy.

Characterization of N-methylcanadine and N-methylstylopine metabolites in rat liver S9 by high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry.

RATIONALE: N-Methylcanadine and N-methylstylopine are two types of isoquinoline alkaloids which are considered to be the main medicinally active constituents of the genus Papaveraceae. However, to date, no metabolism studies of N-methylcanadine and N-methylstylopine have been reported. Therefore, the purpose of the present study was to investigate the in vitro metabolism of these two alkaloids in rat liver S9. METHODS: N-Methylcanadine or N-methylstylopine was incubated with rat liver S9 for 1 h, and then the incubation mixture was processed with 15% trichloroacetic acid. High-performance liquid chromatography with quadrupole time-of-flight mass spectrometry (HPLC/QqTOF-MS) as a reliable analytical method was used. The structural characterization of these metabolites was performed by the combination of the accurate MS/MS spectra and the known elemental composition. RESULTS: As a result, a total of four metabolites of N-methylcanadine and five metabolites of N-methylstylopine in rat liver S9 were tentatively identified. The cleavage of the methylenedioxy group of the drugs was the main metabolic pathway of N-methylcanadine and N-methylstylopine. CONCLUSIONS: The present study is the first in vitro metabolic investigation of N-methylcanadine and N-methylstylopine in rat liver S9 using a reliable HPLC/QqTOF-MS method. The metabolic pathways of N-methylcanadine and N-methylstylopine are tentatively proposed. This work lays the foundation for the in vivo metabolism of the two compounds in animals.

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.

Safety of standardized Macleaya cordata extract in an eighty-four-day dietary study in dairy cows.

The objective of this study was to evaluate the safety of a standardized Macleaya cordata Extract Product (MCEP) containing the quaternary benzophenanthridine alkaloids, sanguinarine and chelerythrine, when fed to dairy cows. Thirty-six dairy cows were randomized into three groups with twelve cows/treatment in two replica pens for each treatment group: control (C) without MCEP added to feed, treatment 1 (SANG-1000) with MCEP added to feed at 1,000 mg/animal/day (1.5 mg/kg bw/day) and treatment 2 (SANG-10000) with MCEP added to feed at 10,000 mg/animal/day (15.5 mg MCEP/kg bw/day). After two weeks of acclimation, animals were observed for an 84-day experimental period, with body weight, feed intake and milk production measured daily. Milk composition was analysed every two weeks. Haematological analyses were performed on Day 0 and Day 84, and clinical chemistry analyses were performed on Day 84 of the study. There was no statistically significant difference (p > .10) among the three groups on body condition score, milk production or milk composition over the study period. There were no significant differences in body weight gain or feed consumption among the three groups. Animals in the SANG-10000 group had significantly higher mean corpuscular volume (MCV) than the C group (p < .1) and lower mean corpuscular haemoglobin concentration (MCHC) than the SANG-1000 group (p < .1). Concentrations of sanguinarine and chelerythrine in milk samples collected on Day 84 were below the detection limit (LOD) as measured by high-performance liquid chromatography-mass spectrometry (HPLC-MS/MS). In conclusion, this study presents compelling data supporting the hypothesis that the test product MCEP, when included in the TMR at up to 10,000 mg/animal/day (15.5 mg MCEP/kg bw/day), is well tolerated by dairy cows.

The impacts of Macleaya cordata extract and naringin inclusion in post-weaning piglet diets on performance, nutrient digestibility and intestinal histomorphology.

The current study investigated the impacts of supplementation of post-weaning piglet diets with Macleaya cordata extract (MCE) and naringin (NAR) on performance, nutrient digestibility and intestinal histomorphology. Post-weaning crossbred piglets (28 males and 28 females; age at weaning 25 d) were randomly allotted to 28 pens. The experiment consisted of a control and three treatment groups (MCE60, MCE120 and NAR). For diets MCE60 and MCE120, the control diet was supplemented with 60 and 120 mg Sangrovit® Extra (a standardised premixture of MCE) per kg diet, respectively. Group NAR received 50 mg pure NAR per kg diet. The experiment lasted 42 d (d 25 - 66 of age). At d 66, apparent pre-caecal digestibility (APD) of nutrients was determined and histomorphological changes in mid-jejunum were evaluated. Feeding diets MCE120 and NAR improved body weight gain and feed conversion ratio of piglets. After feeding diets MCE120 and NAR, the APD of phosphorus and different single and total amino acids were greater than after feeding the control diet. The present data demonstrated that supplementation of post-weaning piglet diets with 120 mg MCE or 50 mg NAR per kg diet could improve growth performance and nutrient digestibility and had no impact on histomorphological variables in the jejunum. These findings indicate the potential of these products to be used as growth promoters in pig nutrition.

Tissue-specific metabolite profiling of benzylisoquinoline alkaloids in the root of Macleaya cordata by combining laser microdissection with ultra-high-performance liquid chromatography/tandem mass spectrometry.

RATIONALE: Tissue-specific metabolite profiling helps to find trace alkaloids masked during organ analysis, which contributes to understanding the alkaloid biosynthetic pathways in vivo and evaluating the quality of medical plants by morphology. As Macleaya cordata contains diverse types of benzylisoquinoline alkaloids (BIAs), the alkaloid metabolite profiling was carried out on various tissues of the root. METHODS: Laser microdissection with fluorescence detection was used to recognize and dissect different tissues from the root of M. cordata. Ultra-high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry was applied to analyze the trace alkaloids in tissues. These detected alkaloids were elucidated using their accurate molecular weights, MS/MS data, MS fragmentation patterns and the known biosynthetic pathways of BIAs. Finally, the distribution of alkaloids in dissected tissues and whole sections was mapped. RESULTS: Forty-nine alkaloids were identified from five microdissected tissues, and 24 of them were detected for the first time in M. cordata. Some types of alkaloids occurred specifically in dissected tissues. More alkaloids were detected in the cork and xylem vascular bundles which emit strong fluorescence under fluorescence microscopy. Some of the screened alkaloids were intermediates in sanguinarine and chelerythrine biosynthetic pathways, and others were speculated to be involved in the new branches of biosynthetic pathways. CONCLUSIONS: The integrated method is sensitive, specific and reliable for determining trace alkaloids, which is also a powerful tool for metabolite profiling of tissue-specific BIAs in situ. The present findings should contribute to a better understanding of the biosynthesis of BIAs in M. cordata root and provide scientific evidence for its quality evaluation based on morphological characteristics. Copyright © 2016 John Wiley & Sons, Ltd.