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.

Structurally Diverse Bisbenzylisoquinoline Alkaloids with Antiadipogenic Activity through PPARγ Downregulation from the Embryo of Nelumbo nucifera Seeds.

Six undescribed and six known bisbenzylisoquinoline alkaloids were isolated from the embryo of Nelumbo nucifera seeds. Their structures were fully characterized by a combination of 1H, 13C NMR, 2D NMR, and HRESIMS analyses, as well as ECD computational calculations. The antiadipogenic activity of 11 alkaloids was observed in a dose-responsive manner, leading to the suppression of lipid accumulation in 3T3-L1 cells. Luciferase assay and Western blot analysis showed that the active alkaloids downregulated peroxisome proliferator-activated receptor gamma (PPARγ, a key antiadipogenic receptor) expression in 3T3-L1 cells. Analysis of the structure-activity relationship unveiled that a 1R,1'S configuration in bisbenzylisoquinoline alkaloids led to a notable enhancement in antiadipogenic activity. The resistance level against lipid accumulation highlighted a consistent pattern with the suppressive effect on the PPARγ expression. These activity results indicate that alkaloids from the embryo of N. nucifera seeds have a potential of antiobesity effects through PPARγ downregulation.

Seco-neferine A-F, three new pairs of benzyltetrahydroisoquinoline alkaloid epimers from Plumula Nelumbinis and their activity.

Three new pairs of benzyltetrahydroisoquinoline (BIQ) alkaloid epimers, Seco-neferine A-F (1-6), were isolated from an EtOH extract of Plumula Nelumbinis. The structures of these compounds were identified by a combination of NMR, HR-ESI-MS, circular dichroism, UV spectroscopic analyses and specific rotations. The structure of compounds 1-6 possesses high similarity with neferine, because these three pairs of epimers have the same skeleton as neferine. Compounds 1,2 and 5,6 are open-loop compounds of position 1' and 1 of neferine respectively. The H connects with position 2' N of compounds 1,2 is replaced by methyl, forming the structure of compounds 3,4. Moreover, six compounds were tested for cytotoxicity against MDA-MB-231 breast cancer cell. Compound 6 displayed moderate inhibitory effects on breast cancer with IC50 of 38.96 µM, while compounds 2,3,4 show certain inhibitory effects.

Analgesic bisbenzylisoquinoline alkaloids from the rhizoma of Menispermum dauricum DC.

Fifteen new bisbenzylisoquinoline alkaloids (1-15) were isolated from the rhizome of Menispermum dauricum DC. Compounds 1-9 were new N-oxides of dauricine-type alkaloids. Compounds 10-14 were rare tail-to-tail quaternary alkaloids. Their structures were characterized by comprehensive analysis of spectroscopic data, and absolute configurations were established from electronic circular dichroism (ECD) data and ECD calculations. Compounds were assayed on analgesic-related G-protein coupled receptors (GPCRs) including dopamine D1 and D2 receptors, opioid Mu receptor and muscarinic M3 receptor. Compound 1 showed high affinity and selective antagonistic activity on the M3 receptor with an IC50 value of 2.2 ± 0.5 µM; compound 15 exhibited the highest antagonistic affinity among the evaluated compounds on Mu (IC50 = 1.1 ± 0.6 µM) and it also acted as a D1 receptor antagonist (IC50 = 8.8 ± 2.9 µM). These findings expanded the existing library of bisbenzylisoquinoline alkaloids and provided new structures for the related future drug design and synthesis.

The Cholinesterase Inhibitory Properties of Stephaniae Tetrandrae Radix.

Stephaniae tetrandrae radix (STR) is a commonly used traditional Chinese medicine in alleviating edema by inducing diuresis. In the clinic, STR extracts or its components are widely used in the treatment of edema, dysuria, and rheumatism for the regulation of water metabolism. Furthermore, STR has been used in treating emotional problems for years by combining with other Chinese herbs. However, the material basis and mechanism of STR on the nervous system have not been revealed. Here, the main components of STR extracts with different extracting solvents were identified, including three major alkaloids, i.e., cyclanoline, fangchinoline, and tetrandrine. The cholinesterase inhibitory activity of STR extracts and its alkaloids was determined using the Ellman assay. Both cyclanoline and fangchinoline showed acetylcholinesterase (AChE) inhibitory activity, demonstrating noncompetitive enzyme inhibition. In contrast, tetrandrine did not show enzymatic inhibition. The synergism of STR alkaloids with huperzine A or donepezil was calculated by the median-effect principle. The drug combination of fangchinoline-huperzine A or donepezil synergistically inhibited AChE, having a combination index (CI) < 1 at Fa = 0.5. Furthermore, the molecular docking results showed that fangchinoline bound with AChE residues in the peripheral anionic site, and cyclanoline bound with AChE residues in the peripheral anionic site, anionic site, and catalytic site. In parallel, cyclanoline bound with butyrylcholinesterase (BChE) residues in the anionic site, catalytic site, and aromatic site. The results support that fangchinoline and cyclanoline, alkaloids derived from STR, could account for the anti-AChE function of STR. Thus, STR extract or its alkaloids may potentially be developed as a therapeutic strategy for Alzheimer's patients.

Neferine, a bisbenzylisoquinoline alkaloid of Nelumbo nucifera, inhibits glutamate release in rat cerebrocortical nerve terminals through 5-HT1A receptors.

Neferine, a bisbenzylisoquinoline alkaloid present in Nelumbo nucifera, has been reported to exhibit neuroprotective effects. Because reduced glutamatergic transmission through inhibition of glutamate release has been proposed as a mechanism of neuroprotection, we investigated whether and how neferine inhibits glutamate release in the nerve terminals of the cerebral cortex of rats. The results demonstrated that neferine inhibits the glutamate release that is evoked by the potassium channel blocker 4-aminopyridine, doing so in a dose-dependent manner. This effect was prevented by removing extracellular calcium and blocking vesicular transporters or N- and P/Q-type calcium channels but not by blocking glutamate transporters. Neferine decreased the 4-aminopyridine-stimulated elevation in intrasynaptosomal calcium concentration; however, it had no effect on the synaptosomal membrane potential. The inhibition of glutamate release by neferine was also eliminated by the selective 5-hydroxytryptamine 1A (5HT1A) receptor antagonist WAY100635, Gi/o protein inhibitor pertussis toxin, adenylyl cyclase inhibitor MDL12330A, and protein kinase A inhibitor H89. Moreover, immunocytochemical analysis revealed the presence of 5-HT1A receptor proteins in the vesicular transporter of glutamate type 1 positive synaptosomes. The molecular docking study also demonstrated that neferine exhibited the highest binding affinity with 5-HT1A receptors (Autodock scores for 5-HA1A = -11.4 kcal/mol). Collectively, these results suggested that neferine activates 5-HT1A receptors in cortical synaptosomes, which decreases calcium influx and glutamate release through the activation of Gi/o protein and the inhibition of adenylyl cyclase/cAMP/protein kinase A cascade.

Speed of action and stage specificity of Bencha-loga-wichian, a Thai traditional antipyretic formulation, against Plasmodium falciparum and the chloroquine-potentiating activity of its active compounds, tiliacorinine and yanangcorinine.

ETHNOPHARMACOLOGICAL RELEVANCE: Bencha-loga-wichian (BLW), a Thai traditional antipyretic formulation, has been reported to have promising antiplasmodial activity, and it was previously revealed that tiliacorinine and yanangcorinine, isolated from Tiliacora triandra, were the active compounds. However, the mechanisms of action of BLW have not been investigated. In addition, these active compounds are bisbenzylisoquinoline alkaloids, many compounds of which have been reported to potentiate the efficacy of chloroquine. AIMS OF THE STUDY: To investigate the antiplasmodial mechanisms of action of BLW and evaluate the effects of chloroquine combined with tiliacorinine or yanangcorinine. MATERIALS AND METHODS: Chloroquine-resistant Plasmodium falciparum (PfW2) strains at the ring, trophozoite, and schizont stages were exposed to the extracts or compounds for 2, 4, 6, 8, 10, 12, 24 or 48 h. The percentages of parasitemia were determined by flow cytometry, and their morphologies were examined by Giemsa-stained smear to evaluate the speed of action and stage specificity. For the drug combination assay, a modified fixed-ratio isobologram method was used. RESULTS: The antiplasmodial activity of BLW possessed a slow onset of action and was the most effective against ring-stage parasites. After 48 h of extracts or compounds exposure, most of the treated parasites, at all stages, turned to the pyknotic form and could not recover even after extracts or compounds removal. The results suggested that these extracts and compounds could kill the parasites or possess parasiticidal effects. In addition, the combination of chloroquine with tiliacorinine or yanangcorinine demonstrated a synergistic effect, indicating that these compounds could potentiate chloroquine efficacy against chloroquine-resistant parasites. CONCLUSION: The antiplasmodial mechanisms of action of BLW appeared to differ from that of chloroquine and other current antimalarial drugs. In addition, tiliacorinine and yanangcorinine, the active compounds of BLW, could potentiate the efficacy of chloroquine. Accordingly, BLW was shown to be a good candidate for development as a new antimalarial and useful for drug combination therapy.

In vitro inhibitory effects of cepharanthine on human liver cytochrome P450 enzymes.

Context: Cepharanthine (CEP) extracted from the roots of Stephania cepharantha Hayata (Menispermaceae), has a range of therapeutic potential in clinical conditions. Whether it affects the activity of human liver cytochrome P450 (CYP) enzymes remains unclear.Materials and methods: The effects of CEP (100 µM) on eight human liver CYP isoforms (i.e., 1A2, 3A4, 2A6, 2E1, 2D6, 2C9, 2C19 and 2C8) were investigated in vitro using human liver microsomes (HLMs) with specific probe actions and probe substrates. In addition, the enzyme kinetic parameters were calculated.Results: The results showed that the activity of CYP3A4, CYP2E1 and CYP2C9 was inhibited by CEP, with IC50 values of 16.29, 25.62 and 24.57 µM, respectively, but other CYP isoforms were not affected. Enzyme kinetic studies showed that CEP was not only a non-competitive inhibitor of CYP3A4 but also a competitive inhibitor of CYP2E1 and CYP2C9, with Ki values of 8.12, 11.78 and 13.06 µM, respectively. Additionally, CEP is a time-dependent inhibitor for CYP3A4 with KI/Kinact value of 10.84/0.058 min/µM.Discussion and conclusions: The in vitro studies of CEP with CYP isoforms indicate that CEP has the potential to cause pharmacokinetic drug interactions with other co-administered drugs metabolized by CYP3A4, CYP2E1 and CYP2C9. Further clinical studies are needed to evaluate the significance of this interaction.

Thalictrum foliolosum: A lesser unexplored medicinal herb from the Himalayan region as a source of valuable benzyl isoquinoline alkaloids.

ETHNOPHARMACOLOGICAL RELEVANCE: Thalictrum foliolosum DC (Ranunculaceae) is a perennial flowering herb traditionally used as a tonic, antiperiodic, diuretic, febrifuge, purgative and stomachic and for the treatment of snakebite, jaundice, and rheumatism. AIM OF THE STUDY: To provide a critical assessment of the state-of-the-art related to the traditional uses, phytochemistry, and pharmacology of T. foliolosum with the ultimate objective of providing further research strategies to facilitate the exploitation of the therapeutic potential of T. foliolosum for the treatment of human disorders. MATERIALS AND METHODS: Exhaustive bibliographic research related to T. foliolosum plant was carried out using scientific research engines and databases such as Google Scholar, PubMed, Web of Science covering all retrieved relevant manuscripts written in English. RESULTS: Several alkaloids such as berberine, jatrorrhizine, palmatine, thalrugosidine, thalrugosaminine, thalisopine (thaligosine), thalirugidine, thalirugine, 8-oxyberberine (berlambine), noroxyhydrastinine, N,O,O-trimethylsparsiflorine, thalicarpine, thalidasine, thalfoliolosumines A and thalfoliolosumines B were reported from T. foliolosum. Ethnomedicinal studies revealed much wider scope of T. foliolosum in developing various drugs to solve multiple challenges in the health sector. Therapeutic effects were attributed to the bioactivities of the secondary metabolites present in T. foliolosum. CONCLUSIONS: T. foliolosum is rich in berberine and other benzylisoquinoline alkaloids. T. foliolosum can be used as an excellent and effective herbal remedy for various human ailments since there are no reports on the toxicity of this herb.

Bioactive bisbenzylisoquinoline alkaloids from the roots of Stephania tetrandra.

Ten new bisbenzylisoquinoline alkaloids (1-10) and eight known analogues (11-18) were obtained from the roots of Stephania tetrandra. The structures of these compounds were determined by spectroscopic methods, single-crystal X-ray diffraction, electronic circular dichroism analyses, and chemical method. Compounds 1, 15, and 16 showed the better anti-inflammatory activities with IC50 values of 15.26 ± 2.99, 6.12 ± 0.25, and 5.92 ± 1.89 µM, respectively. Compound 18 possessed cytotoxic activities against MCF-7, HCT-116, and HepG2 cell lines with IC50 values of 2.81 ± 0.06, 3.66 ± 0.26, and 2.85 ± 0.15 µM, respectively.

Phthalideisoquinoline Hemiacetal Alkaloids from Corydalis decumbens That Inhibit Spontaneous Calcium Oscillations, Including Alkyl Derivatives of (+)-Egenine That Are Strikingly Levorotatory.

The new phthalideisoquinoline hemiacetal alkaloids (2-7) and the known analogues (1 and 8) were isolated from the bulbs of Corydalis decumbens. The new compounds were characterized by analysis of their NMR spectroscopic data, chemical degradation syntheses, X-ray crystallography, and comparison of experimental and calculated ECD data. All the isolates were screened in vitro for inhibitory activity of spontaneous calcium oscillations in primary cultured neocortical neurons. Compounds 1-3 and 5-7 were found to be active in the suppression of spontaneous calcium oscillations with IC50 values of 6.8, 5.6, 11.6, 10.2, 8.3, and 3.1 µM, respectively. It was also observed that the presence of hydroxy, methoxy, and ethoxy groups at the remote stereogenic center C-7' of some isolated phthalideisoquinoline hemiacetal alkaloids could alter the preferred conformation and invert the sign of optical rotation, rather than this resulting from configurational isomerism at C-1 or C-9, and that the 3J1,9 coupling constants of these analogues varied accordingly. For example, compounds 1 and 6 are levorotatory, despite these molecules having the same carbon skeleton and absolute configuration as (+)-egenine. This emphasizes the potential risk of incorrectly assigning absolute configuration based only on observed coupling constants or optical rotation when comparing the data of new compounds with literature values for known analogues, especially within this class of molecules.

Molecular mechanisms involved in drug-induced liver injury caused by urate-lowering Chinese herbs: A network pharmacology study and biology experiments.

As an important part of the comprehensive treatment methods, the urate-lowering Chinese herbs could provide favorable clinical effects on hyperuricemia in its ability to invigorate spleen and remove dampness. Owing to the long-term duration, it brought up the potential adverse reactions (ADRs) and concerns about the drug-induced liver injury from these herbs. To address this problem, the bioinformatics approaches which combined the network pharmacology, computer simulation and molecular biology experiments were undertaken to elucidate the underlying drug-induced liver injury molecular mechanisms of urate-lowering Chinese herbs. Several electronic databases were searched to identify the potential liver injury compounds in published research. Then, the putative target profile of liver injury was predicted, and the interaction network was constructed based on the links between the compounds, corresponding targets and core pathways. Accordingly, the molecular docking simulation was performed to recognize the representative compounds with hepatotoxicity. Finally, the cell experiments were conducted to investigate the biochemical indicators and expression of the crucial protein that were closely associated with liver injury. In conclusion, the current research revealed that the compounds with potential liver injury including diosgenin, baicalin, saikosaponin D, tetrandrine, rutaecarpine and evodiamine from urate-lowering Chinese herbs, could lead to decline the survival rate of L-02 cell, increase the activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) in cell-culture medium, enhance the expression of p-p38/p38, while the p38 inhibitor could achieve the trend of regulating and controlling liver injury. These research findings bring further support to the growing evidence that the mechanism of the liver injury induced by the compounds from urate-lowering Chinese herbs may be associated with the activation of p38α.

Optimization of column chromatography technique for the isolation of tetrandrine from Cyclea peltata and LC-ESI-MS based quantification and validation of the method.

Reverse phase column chromatography technique was employed to isolate tetrandrine from methanolic extract of Cyclea peltata roots. Tetrandrine was efficiently isolated using 0.02% aqueous diethylamine and methanol (25:75, v/v) as mobile phase with a purity of 98.63%. Compound characterisation was achieved using high-performance liquid chromatography coupled with electrospray ionization mass spectrometry and other spectroscopic methods. In addition, quantification of tetrandrine and validation of the method was carried out following International Conference on Harmonisation guidelines. The study provides a simple and cost effective method to isolate substantially good quantity and pure tetrandrine from Cyclea peltata.

Bisbenzylisoquinoline Alkaloids.

The present review covers the literature on bisbenzylisoquinoline alkaloids from 1999 through early 2018. About 500 natural products belong to this large alkaloid class, of which 65 were characterized for the first time in the period covered by this chapter. The review comprises a classification of bisbenzylisoquinoline alkaloids and gives an overview of the principal biosynthetic pathways. Furthermore, a list of the novel natural products as well as their available analytical data are included in this chapter. Special emphasis is given to the progress in the total synthesis of bisbenzylisoquinoline alkaloids. To accommodate the drastically increased number of publications concerning the biological and pharmacological evaluation, an overview on the most important biological findings is provided.

Quantification of natural products in herbal supplements: A combined NMR approach applied on goldenseal.

Authentication of natural products is of major relevance in the context of manufactured drugs or herbal supplements since such active products generate a lucrative market. The analytical method to identify and quantify valuable natural products is critical for quality control and product assignment of herbal supplements. In this framework, we propose to apply a recently developed quantitative 2D NMR approach called Q QUIPU (Quick QUantItative Perfected and pUre shifted) in combination with 1D 1H NMR capable to access the concentration of three major alkaloids, berberine, ß-hydrastine and canadine, in the root extract of goldenseal (Hydrastis canadensis), one of the 20 most popular herbal supplements used worldwide. We highlight the complementarity of 1D and 2D quantitative NMR to accurately assess the amount of alkaloids with different range of concentrations and stability within extracts. In particular, unstable natural products having non-overlapped signals like berberine could only be quantified by sensitive and fast 1D 1H, while overlapped signals of ß-hydrastine and low intense ones of canadine could only be quantified with the recent 2D Q QUIPU HSQC. Results obtained from this combined approach have led to a good accuracy (<10%) as compared with coupled UHPLC-MS/UV techniques. This quantitative NMR approach paves the way to numerous applications where the accurate quantification of targeted compounds in complex mixtures is required, for instance in agricultural, food and pharmaceuticals products.

Neferine, a Bisbenzylisoquinoline Alkaloid, Ameliorates Dextran Sulfate Sodium-Induced Ulcerative Colitis.

Both the incidence and prevalence of ulcerative colitis (UC) are increasing throughout the world. Neferine, a natural alkaloid, demonstrated a variety of biological activities. In this study, the anti-inflammatory effect of neferine was investigated. Raw264.7 cells were stimulated with lipopolysaccharide (LPS) or LPS plus Z-VAD-fmk (Z-VAD). The inhibitory effect of neferine on secretion of nitrite, cytokines tumor necrosis factor alpha (TNF-[Formula: see text]) and interleukin 6 (IL-6), expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) was determined. The protective effect of neferine was investigated in dextran sulfate sodium (DSS)-induced UC mouse model. Neferine significantly inhibited LPS and LPS plus Z-VAD induced secretion of nitrite, cytokines, and expression of iNOS and COX-2. Oral administration of neferine (10[Formula: see text]mg/kg and 25[Formula: see text]mg/kg) significantly reduced DSS-induced mouse weight loss, decreased disease activity index (DAI) scores, improved colon pathological changes, and decreased plasma cytokines. In addition, neferine significantly inhibited the protein expression of iNOS, COX-2, receptor-interacting protein 1 (RIP1), RIP3, mixed lineage kinase domain-like protein (MLKL), and increased the protein expression of caspase-8 in colon tissues. These data suggest that neferine was a potent anti-inflammatory agent against LPS and DSS induced inflammation both in vitro and in vivo.

Dauricine combined with clindamycin inhibits severe pneumonia co-infected by influenza virus H5N1 and Streptococcus pneumoniae in vitro and in vivo through NF-κB signaling pathway.

Dauricine, isolated from Menispermum dauricum, has been widely used for treatment of various diseases, including cardiac ischemia and inflammation-related diseases. However, little is known regarding to the effect of dauricine on severe pneumonia. Therefore, the aim was to investigate the effect of dauricine on severe pneumonia and its mechanism during progress. Herein, H5N1 and Streptococcus pneumoniae (D39) were conducted to induce severe pneumonia in both BEAS-2B cells and mice. In vitro, dauricine reversed the protein and mRNA expressions of TNF-α, IL-6 and IL-1ß, examined by ELISA and qRT-PCR assay, respectively. In addition, the nuclear translocation of NF-κB/p65 and the phosphorylation expressions of IκBα and IKKα/ß, examined by western blotting, were dose-dependently dropped by dauricine. However, dauricine had no significant effect on MAPKs, including JNK, ERK and p38. In vivo, dauricine significantly decreased MPO activity, the lung wet/dry weight ratio, the protein and mRNA expression of TNF-α, IL-6 and IL-1ß, the expressions of NF-κB/p65, and attenuated the lung histological alterations. Besides, compared to dauricine alone, combined with clindamycin had more remarkably effects on severe pneumonia in vitro. Overall, the results suggested that dauricine, a relatively drug that targets NF-κB, in combination with clindamycin, maybe a novel therapeutic strategy for severe pneumonia.

Anticancer activity of tetrandrine by inducing pro-death apoptosis and autophagy in human gastric cancer cells.

OBJECTIVES: To investigate the antitumour property of tetrandrine by inducing autophagy and apoptosis in human gastric cancer cells, and to explore the potential molecular mechanisms. METHODS: The antitumour activity of tetrandrine was assessed through MTT assay. Apoptosis was measured by flow cytometry and microscopic examination of cellular morphology. The mitochondrial membrane potential was detected by staining with Rh-123. Induction of autophagy was monitored by transmission electron microscopy observation, using GFP-LC3 transfection. KEY FINDINGS: The results revealed that tetrandrine exhibits significant antitumour activity against gastric human cancer cell and the antigastric tumour activity was depended on inducing autophagy and apoptosis through upregulating the apoptosis-related protein (cleaved PARP, cleaved caspase-3 and cleaved caspase-9) and autophagy-related protein (Beclin-1, LC3-II and p62), and decreasing the phosphorylation of AKT/mTOR, PS6K and P-4EBP1. Adding the inhibitor of autophagy, 3-MA or Baf-A1, increased the viability of tetrandrine-exposed gastric cancer cells, which confirmed the role of autophagy played in the gastric cancer cell death induced by tetrandrine. CONCLUSIONS: These results demonstrated that the antitumour effects of tetrandrine by inducing autophagy and apoptosis involving Akt/mTOR pathway. Thus, tetrandrine may be a promising lead compound to be further developed in future for cancer therapy.

The norpurpureine alkaloid from Annona purpurea inhibits human platelet activation in vitro.

BACKGROUND: The leaves of Annona purpurea have yielded several alkaloids with anti-aggregation activities against rabbit platelets. This is promising in the search for agents that might act against platelets and reduce the incidence of cardiovascular diseases. Since significant differences in platelet function have been reported between human and animal platelets, a study focusing on the effect of A. purpurea extracts against human platelet activation is necessary. METHODS: The compounds in an A. purpurea ethanolic extract underwent bio-guided fractionation and were used for in vitro human platelet aggregation assays to isolate the compounds with anti-platelet activity. The bioactive compounds were identified by spectroscopic analysis. Additional platelet studies were performed to characterize their action as inhibitors of human platelet activation. RESULTS: The benzylisoquinoline alkaloid norpurpureine was identified as the major anti-platelet compound. The IC50 for norpurpureine was 80 µM against platelets when stimulated with adenosine 5'-diphosphate (ADP), collagen and thrombin. It was pharmacologically effective from 20 to 220 µM. Norpurpureine (220 µM) exhibited its in vitro effectiveness in samples from 30 healthy human donors who did not take any drugs during the 2 weeks prior to the collection. Norpurpureine also gradually inhibited granule secretion and adhesion of activated platelets to immobilized fibrinogen. At the intra-platelet level, norpurpureine prevented agonist-stimulated calcium mobilization and cAMP reduction. Structure-activity relationship analysis indicates that the lack of a methyl group at the nitrogen seems to be key in the ability of the compound to interact with its molecular target. CONCLUSION: Norpurpureine displays a promising in vitro pharmacological profile as an inhibitor of human platelet activation. Its molecular target could be a common effector between Ca2+ and cAMP signaling, such as the PLC-PKC-Ca2+ pathway and PDEs. This needs further evaluation at the protein isoform level.