Peimine, an Anti-Inflammatory Compound from Chinese Herbal Extracts, Modulates Muscle-Type Nicotinic Receptors.

Fritillaria bulbs are used in Traditional Chinese Medicine to treat several illnesses. Peimine (Pm), an anti-inflammatory compound from Fritillaria, is known to inhibit some voltage-dependent ion channels and muscarinic receptors, but its interaction with ligand-gated ion channels remains unexplored. We have studied if Pm affects nicotinic acetylcholine receptors (nAChRs), since they play broad functional roles, both in the nervous system and non-neuronal tissues. Muscle-type nAChRs were incorporated to Xenopus oocytes and the action of Pm on the membrane currents elicited by ACh (IAChs) was assessed. Functional studies were combined with virtual docking and molecular dynamics assays. Co-application of ACh and Pm reversibly blocked IACh, with an IC50 in the low micromolar range. Pm inhibited nAChR by: (i) open-channel blockade, evidenced by the voltage-dependent inhibition of IAch, (ii) enhancement of nAChR desensitization, revealed by both an accelerated IACh decay and a decelerated IACh deactivation, and (iii) resting-nAChR blockade, deduced from the IACh inhibition elicited by Pm when applied before ACh superfusion. In good concordance, virtual docking and molecular dynamics assays demonstrated that Pm binds to different sites at the nAChR, mostly at the transmembrane domain. Thus, Pm from Fritillaria bulbs, considered therapeutic herbs, targets nAChRs with high affinity, which might account for its anti-inflammatory actions.

Peiminine Suppresses RANKL-Induced Osteoclastogenesis by Inhibiting the NFATc1, ERK, and NF-κB Signaling Pathways.

Osteoclasts (OCs) play an important role in osteoporosis, a disease that is mainly characterized by bone loss. In our research, we aimed to identify novel approach for regulating osteoclastogenesis and thereby treating osteoporosis. Previous studies have set a precedent for screening traditional Chinese herbal extracts for effective inhibitors. Peiminine is an alkaloid extracted from the bulb of Fritillaria thunbergii Miq that reportedly has anticancer and anti-inflammatory effects. Thus, the potential inhibitory effect of peiminine on OC differentiation was investigated via a series of experiments. According to the results, peiminine downregulated the levels of specific genes and proteins in vitro and consequently suppressed OC differentiation and function. Based on these findings, we further investigated the underlying molecular mechanisms and identified the NF-κB and ERK1/2 signaling pathways as potential targets of peiminine. In vivo, peiminine alleviated bone loss in an ovariectomized mouse model.

In vitro effects of peimine on the activity of cytochrome P450 enzymes.

Peimine is a major component of Fritillaria ussuriensis, which is a widely used herb in pediatric. It is very common in Chinese traditional medicine to combine with two or more herbs in the clinic. To investigate the effect of peimine on the activity of cytochrome P450 enzymes (CYP450) is necessary for the clinical application of peimine.The effects of peimine on eight human liver CYP isoforms (i.e., 1A2, 3A4, 2A6, 2E1, 2D6, 2C9, 2C19 and 2C8) were investigated in vitro in human liver microsomes (HLMs) with the specific inhibitors as positive control and without peimine or inhibitors as negative control. The enzyme kinetic parameters were calculated.It was found that peimine inhibited the activity of CYP3A4, 2E1, and 2D6 in a concentration-dependent manner with the IC50 values of 13.43, 21.93, and 22.46 µM, respectively. The inhibition of CYP3A4 was performed in a non-competitive manner with the Ki value of 6.49 µM, and the inhibition of CYP2E1 and 2D6 was performed in a competitive manner with Ki values of 10.76 and 11.95 µM. Additionally, peimine inhibited the activity of CYP3A4 in a time-dependent manner with the KI/Kinact value of 6.17/0.049 min-1 µM-1.Peimine inhibited the activity of CYP3A4, 2E1, and 2D6, which indicated the potential interaction between peimine and drugs metabolized by CYP3A4, 2E1, and 2D6. Further studies are needed to verify the drug-drug interaction and the in vivo effects.

Peiminine Inhibits Glioblastoma in Vitro and in Vivo Through Cell Cycle Arrest and Autophagic Flux Blocking.

BACKGROUND/AIMS: Glioblastoma multiforme (GBM) is the most devastating and widespread primary central nervous system tumour in adults, with poor survival rate and high mortality rates. Existing treatments do not provide substantial benefits to patients; therefore, novel treatment strategies are required. Peiminine, a natural bioactive compound extracted from the traditional Chinese medicine Fritillaria thunbergii, has many pharmacological effects, especially anticancer activities. However, its anticancer effects on GBM and the underlying mechanism have not been demonstrated. This study was conducted to investigate the potential antitumour effects of peiminine in human GBM cells and to explore the related molecular signalling mechanisms in vitro and in vivo Methods: Cell viability and proliferation were detected with MTT and colony formation assays. Morphological changes associated with autophagy were assessed by transmission electron microscopy (TEM). The cell cycle rate was measured by flow cytometry. To detect changes in related genes and signalling pathways in vitro and in vivo, RNA-seq, Western blotting and immunohistochemical analyses were employed. RESULTS: Peiminine significantly inhibited the proliferation and colony formation of GBM cells and resulted in changes in many tumour-related genes and transcriptional products. The potential anti-GBM role of peiminine might involve cell cycle arrest and autophagic flux blocking via changes in expression of the cyclin D1/CDK network, p62 and LC3. Changes in Changes in flow cytometry results and TEM findings were also observed. Molecular alterations included downregulation of the expression of not only phospho-Akt and phospho-GSK3ß but also phospho-AMPK and phospho-ULK1. Furthermore, overexpression of AKT and inhibition of AKT reversed and augmented peiminine-induced cell cycle arrest in GBM cells, respectively. The cellular activation of AMPK reversed the changes in the levels of protein markers of autophagic flux. These results demonstrated that peiminine mediates cell cycle arrest by suppressing AktGSk3ß signalling and blocks autophagic flux by depressing AMPK-ULK1 signalling in GBM cells. Finally, peiminine inhibited the growth of U251 gliomas in vivo. CONCLUSION: Peiminine inhibits glioblastoma in vitro and in vivo via arresting the cell cycle and blocking autophagic flux, suggesting new avenues for GBM therapy.

Peimine, a main active ingredient of Fritillaria, exhibits anti-inflammatory and pain suppression properties at the cellular level.

Fritillaria is one of the most important herbs in Chinese traditional medicine and represents an annual ï¿¥700 million industry. It is often used as an anti-inflammatory, pain relieving and antitussive medicine. However, the mechanisms of these effects are still unclear. Peimine is one of active ingredients of Fritillaria. Using the patch-clamp technique, we profiled the action of Peimine against selected ion channels stably expressed in HEK 293 cell lines. Our data indicated that Peimine was not only able to block the Nav1.7 ion channel but also preferably inhibited the Kv1.3 ion channel. Thus, the study suggested potential mechanisms of Fritillaria as a pain relieving and anti-inflammatory herb.

Imperialine and Verticinone from Bulbs of Fritillaria wabuensis Inhibit Pro-inflammatory Mediators in LPS-stimulated RAW 264.7 Macrophages.

The bulbs of plants belonging to the Fritillaria cirrhosa-group have been used as antitussive and expectorant herbs in traditional Chinese medicine for thousands of years. In this study, we isolated two isomers of verticinone and imperialine, steroidal alkaloids belonging to the cevanine group, from bulbs of Fritillaria wabuensis, which is a part of the Fritillaria cirrhosa group, and investigated their anti-inflammatory effects and relative mechanisms on lipopolysaccharide-stimulated RAW 264.7 macrophages. Our results clearly demonstrate that verticinone or imperialine could dose-dependently inhibit nitric oxide production and also suppress inducible nitric oxide synthase and cyclooxygenase-2 expressions. In addition, verticinone or imperialine suppress the production of pro-inflammatory cytokines in a dose dependent manner, such as tumor necrosis factor-α and interleukin-1ß. The effect of verticinone and imperialine on the activation of nuclear factor-kappaB was also evaluated. The phosphorylation of nuclear factor-kappaB stimulated with LPS is also down-regulated by verticinone or imperialine in a concentration dependent manner, which coincided with the inhibition of phosphorylation forms of inhibitory kappaB-α, a crucial inhibitory factor of nuclear factor-kappaB. Generally, the anti-inflammatory effects and mechanisms of verticinone and imperialine are mediated by the inhibition of the nuclear factor-kappaB activation signaling pathway. According to the results of our researches, verticinone and imperialine may present great potentials to be developed as therapeutics for inflammatory diseases.

Intestinal absorption characteristics of imperialine: in vitro and in situ assessments.

AIM: Imperialine is an effective compound in the traditional Chinese medicine chuanbeimu (Bulbus Fritillariae Cirrhosae) that has been used as antitussive/expectorant in a clinical setting. In this study we investigated the absorption characteristics of imperialine in intestinal segments based on an evaluation of its physicochemical properties. METHODS: Caco-2 cells were used to examine uptake and transport of imperialine in vitro, and a rat in situ intestinal perfusion model was used to characterize the absorption of imperialine. The amount of imperialine in the samples was quantified using LC-MS/MS. RESULTS: The aqueous solubility and oil/water partition coefficient of imperialine were determined. This compound demonstrated a relatively weak alkalinity with a pKa of 8.467±0.028. In Caco-2 cells, the uptake of imperialine was increased with increasing pH in medium, but not affected by temperature. The apparent absorptive and secretive coefficient was (8.39±0.12)×10(-6) cm/s and (7.78±0.09)×10(-6) cm/s, respectively. Furthermore, neither the P-glycoprotein inhibitor verapamil nor Niemann-Pick C1-Like 1 transporter inhibitor ezetimibe affected the absorption and secretion of imperialine in vitro. The in situ intestinal perfusion study showed that the absorption parameters of imperialine varied in 4 intestinal segments (duodenum, jejunum, ileum and colon) with the highest ones in the colon, where a greater number of non-ionized form of imperialine was present. CONCLUSION: The intestinal absorptive characteristics of imperialine are closely related to its physicochemical properties. The passive membrane diffusion dominates the intestinal absorption of imperialine.

[Effect of Peimine on ERCC1 mRNA and LRP Expressions of A549/DDP Multidrug Resistance Cell Line].

OBJECTIVE: To explore the effect of peimine on excision repair cross-complementation 1 (ERCC1) mRNA and lung resistant protein (LRP) expressions in A549/cisplatin (DDP) multidrug resistance (MDR) cell line. METHODS: Lung cancer A549/DDP cells were cultured in vitro.Cells at logarithmic growth phase were divided into 4 groups, i.e., the blank control group, the DDP group, the ligustrazine group (DDP+ligustrazine), the peimine group (DDP + peimine). After 48-h drug action, ERCC1 mRNA expression was detected by RT-PCR and LRP expression detected by cell immunofluorescence. RESULTS: There was no statistical difference in expression levels of ERCC1 mRNA and LRP between the DDP group and the blank control group (P > 0.05). Compared with the DDP group, expression levels of ERCC1 mRNA and LRP obviously decreased in the ligustrazine group and the peimine group (P < 0.05). They were obviously lower in the peimine group than in the ligustrazine group (P < 0.05). CONCLUSIONS: Peimine could reverse MDR of A549/DDP cell line. Its mechanism might be associated with down-regulating ERCC1 mRNA and LRP expression levels.

Peimisine and peiminine production by endophytic fungus Fusarium sp. isolated from Fritillaria unibracteata var. wabensis.

Steroidal alkaloids, as the major biologically active components in Bulbus Fritillariae, possess a variety of toxicological and pharmacological effects on humans. The objective of this work was to determine whether endophytic fungi isolated from fresh bulbs of Fritillaria unibracteata var. wabensis can produce one or more alkaloids like its host plant. Four classical reagents including Wagner's, iodine-potassium iodide, Mayer's and improved Dragendorff's were used for primary screening. Then thin-layer chromatography (TLC) and high performance liquid chromatography-evaporative light scattering detection (HPLC-ELSD) were employed to identify the fermentation products of the selected strains. The results showed that extract from one stain (WBS007) has positive reactions in process of primary screening. A further TLC scan and HPLC-ELSD showed that strain WBS007 had two components with the same TLC relative front (Rf) value and HPLC retention time (RT) as authentic peimisine and peiminine. In addition, strain WBS007 was identified as Fusarium sp. based on phylogenetic analysis of ITS sequences. Thus, strain WBS007 produced the bioactive ingredient peimisine and peiminine, as does its host plant, and could be used for the production of peimisine and peiminine by fermentation.

Steroidal alkaloids from the bulbs of Fritillaria unibracteata.

Two new steroidal alkaloids peimisine-3-O-ß-D-glucopyranoside (1) and puqiedinone-3-O-ß-D-glucopyranoside (3), together with three known compounds peimisine (2), puqiedinone (4), and puqiedine (5), were isolated and characterized from the bulbs of Fritillaria unibracteata. Their structures were fully elucidated by spectroscopic and chemical methods. Compound 1 showed moderate protection effect on neurotoxicity of PC12 cell lines induced by rotenone.

Structural analysis and antitussive evaluation of five novel esters of verticinone and bile acids.

Shedan-Chuanbei powder, a complex of traditional Chinese medicine preparation, which consists of Snake Bile (Chinese name "Shedan") and Fritillariae Cirrhosae (Chinese name "Chuanbei"), is the most popular antitussive and expectorant formulation in Chinese communities. However, the clinical application of Shedan-Chuanbei powder is now stringently limited because of the shortage of the two crude medicinal materials, especially for the sake of animal protection. In addition, the inherent defects of the most of the complex of traditional Chinese medicine such as the indistinct basal pharmacodynamic materials and the difficulties in quality control had blocked them heading into the international medicinal market. So we attempted to seek new substitute for Shedan-Chuanbei powder for antitussive drugs. In order to gain some new compounds with better bioactivity and attenuated toxicity, we tried to combine two kinds of drugs through ester bond. Enlightened with "combination principle" in drug discovery, we synthesized five novel esters of verticinone and bile acids, both of which are the major bioactive components in Shedan-Chuanbei powder. We then evaluated the antitussive activity and the acute toxicity of the five ester-linked compounds. The five ester-linked compounds had much more potent antitussive activity and expectorant activity than single bile acids at the same doses, and had equivalent antitussive activity and expectorant activity in comparison with about double moles dose of the monomer verticinone. Especially, cholic acid-verticinone ester had much more potent antitussive effects than the monomer verticinone or cholic acid at the same dose. A further acute toxicity study showed that the LD(50) values of the five ester-linked compounds exceeded 3.5g/kg by intraperitoneal injection in mice. Based on the studies of pharmacology and acute toxicity, the five ester-linked compounds have synergic pharmacodynamic action and attenuated toxicity compared with single verticinone and single bile acids.

Verticinone induces cell cycle arrest and apoptosis in immortalized and malignant human oral keratinocytes.

Although verticinone, a major alkaloid isolated from the bulbus of Fritillaria ussuriensis, has been shown to induce differentiation in human leukemia cells, the exact mechanism of this action is not completely understood in cancer cells. Verticinone was used to conduct growth and apoptosis-related experiments for two stages of oral cancer on immortalized human oral keratinocytes (IHOKs) and primary oral cancer cells (HN4). The procedures included MTT assay, three-dimensional (3-D) raft cultures, Western blotting, cell cycle analysis, nuclear staining and cytochrome c expression related to the apoptosis signaling pathway. Verticinone inhibited the proliferation of immortalized and malignant oral keratinocytes in a dose- and time-dependent manner. In 3-D organotypic culture, verticinone-treated cells were less mature than the control cells, displaying low surface keratinization and decreased epithelial thickness. The major mechanism by which verticinone inhibits growth appears to be induced apoptosis and G(0)G(1) cell cycle arrest. This finding is supported by the results of the cell cycle analysis, FITC-Annexin V staining, DNA fragmentation assay and Hoechst 33258 staining. Furthermore, the cytosolic level of cytochrome c was increased, while the expression of Bcl-2 protein was gradually down-regulated and Bax was up-regulated, accompanied by caspase-3 activation. The data suggests that verticinone may induce apoptosis through a caspase pathway mediated by mitochondrial damage in immortalized keratinocytes and oral cancer cells.

Synthesis and antitussive evaluation of verticinone-cholic acid salt, a novel and potential cough therapeutic agent.

AIM: To seek a novel and potent antitussive drug based on Shedan-Chuanbei powder, a complex of traditional Chinese medicine preparation for cough therapy. METHODS: Verticinone-cholic acid (Ver-CA) salt, a novel, salifying derivative of verticinone and cholic acid, both of which are the major bioactive components in Shedan-Chuanbei powder, was synthesized. We then evaluated the antitussive activity and the acute toxicity of the salt. RESULTS: The new compound, with good solubility in water, has much more potent antitussive activity in comparison with the same dose of single verticinone and single cholic acid. The administration 3 mg/kg of Ver-CA could result in over 50% reduction of a citric acid-induced cough. Pretreatment with naloxone (0.8 mg/kg, ip) can only partially antagonize its antitussive effect. On the other hand, glybenclamide (3 mg/kg, ip), an ATP-sensitive K+ channel blocker, can also significantly reduce the antitussive effect of Ver-CA. A further acute toxicity study showed that the LD(50) values of Ver-CA were 3 times that of verticinone. CONCLUSION: Based on the studies of pharmacology and acute toxicity, the salt has a synergic and attenuated toxicity compared with single verticinone and cholic acid. Moreover, the present study also suggests that Ver-CA, a potential novel antitussive agent, may exert its antitussive effect via both the peripheral (modulated by ATP-sensitive K+ channels) and central mechanisms (modulated by the opioid receptor).

[Preparation and antitussive, expectorant and antiasthmatic activities of verticinone-bile acids salts].

To search for potential drugs with potent antitussive, expectorant, antiasthmatic activities and low toxicity, a series of verticinone-bile acids salts were prepared based on the clearly elucidated antitussive, expectorant and antiasthmatic activities of verticinone in bulbs of Fritillaria and different bile acids in Snake Bile. The antitussive, expectorant and antiasthmatic activities of these verticinone-bile acid salts were then screened with different animal models. Ver-CA (verticinone-cholic acid salt) and Ver-CDCA (verticinone-chenodeoxycholic acid salt) showed much more potent activities than other compounds. The bioactivities of Ver-CA and Ver-CDCA are worthy to be intensively studied, and it is also deserved to pay much attention to their much more potent antitussive effects than codeine phosphate. In order to elucidate whether they have synergistic effect and attenuated toxicity, their activities will be continuously compared with single verticinone, cholic acid and chenodeoxycholic acid at the same doses on different animal models. The application of "combination principles" in traditional Chinese medicinal formulations may be a novel way in triditional Chinese medicine research and discovery.

The effects of five alkaloids from Bulbus Fritillariae on the concentration of cAMP in HEK cells transfected with muscarinic M(2) receptor plasmid.

The aim of this study was to investigate the effects of five alkaloids, namely verticine, verticinone, imperialine, imperialine-3beta-D-glucoside, and puqietinone, purified from Bulbus Fritillariae and used as an antitussive drug in traditional Chinese medicine, on their antimuscarinic M(2) function and the cAMP level of HEK cells transfected with muscarinic M(2) receptor plasmid. By transfecting the HEK cells with the method of calcium phosphate co-precipitation and screening with G418, the cells stably expressing M(2) receptor were identified. The expression of M(2) receptor in HEK cells was confirmed by both RT-PCR and western blot. The cAMP level in the treated cells was analyzed with RIA method ((125)I-cAMP KIT). And the results suggested that the five alkaloids could significantly elevate the cAMP concentration in the HEK cells transfected with muscarinic M(2) receptor plasmid (p < 0.01).

Inhibitors of acetylcholine esterase in vitro--screening of steroidal alkaloids from Fritillaria species.

18 alkaloids were successfully isolated from five Fritillaria species and 5 derivatives were synthesized. Their effects on the bioactivity of human whole blood cholinesterase (ChE) were assessed. The results showed that N-demethylpuqietinone, hupeheninoside, ebeiedinone, yibeinoside A and chuanbeinone inhibited the bioactivity of human whole blood ChE at the concentration of 1.0 x 10 ( - 4) M, with the inhibitory effects of 55.5 +/- 2.7 %, 66.8 +/- 2.0 %, 69.0 +/- 1.7 %, 71.2 +/- 1.8 % and 70.7 +/- 3.3 %, respectively. The effects of the five alkaloids on human red blood cell (RBC) acetylcholinesterase (AChE) and human plasma butyrylcholinesterase (BChE) were further studied, and their IC (50) values for human RBC AChE were 6.4 +/- 0.003 microM, 16.9 +/- 0.018 microM, 5.7 +/- 0.004 microM, 6.5 +/- 0.013 microM and 7.7 +/- 0.001 microM, respectively, and the IC50 values for human plasma BChE were 12.5 +/- 0.026 microM, 2.1 +/- 0.005 microM, 5.2 +/- 0.002 microM, 7.3 +/- 0.005 microM and 0.7 +/- 0.001 microM, respectively. These data suggest, therefore, that N-demethylpuqietinone, hupeheninoside, ebeiedinone, yibeinoside A and chuanbeinone have both anti-RBC AChE and anti-plasma BChE activities, N-demethylpuqietinone is a selective inhibitor of AChE, whereas hupeheninoside and chuanbeinone are the selective inhibitors of BChE.

Angiotensin converting enzyme (ACE) inhibitory alkaloids from Fritillaria ussuriensis.

Bioassay-guided fractionation of the BuOH-soluble extract of Fritillaria ussuriensis afforded verticinone ( 1), verticine ( 2), and peimisine ( 3). Purification of these compounds was achieved with the use of various chromatographic methods. The structures of the compounds were identified on the basis of MS and NMR data analysis. Compounds 1 - 3 inhibited angiotensin I converting enzyme activity in a dose-dependent manner, displaying 50 % inhibitory concentration values of 165.0 microM, 312.8 microM, 526.5 microM, respectively. The presence of these active substances may be responsible, at least in part, for the antihypertensive action of the bulbs of Fritillaria ussuriensis.

Differentiation-inducing effects of verticinone, an isosteroidal alkaloid isolated from the bulbus of Fritillaria ussuriensis, on human promyelocytic leukemia HL-60 cells.

The inducer of differentiation of human promyelocytic leukemia HL-60 cells is commonly accepted to have potential therapeutic importance. Verticinone, one of the major isosteroidal alkaloids from the bulbus of Fritillaria ussuriensis, was found to inhibit the growth of HL-60 cells by inducing these cells to differentiate toward granulocytes. Importantly, the combination of verticinone with all-trans retinoic acid (ATRA), a well-known inducer of HL-60 cells into granulocytic lineages, was more effective than either alone, suggesting its therapeutic use in minimizing the effective dose of ATRA.

Structure-activity relationships of imperialine derivatives and their anticholinergic activity.

In order to check the structure-activity relationship and prepare more potent derivatives of imperialine with anticholinergic activity, imperialinol (2), 3 beta-acetoxyimperialine (3), 3 beta-propionoxyimperialine (4), and 3 beta-butyroxyimperialine (5) were prepared. Compounds 4 and 5 displayed better anticholinergic activity against muscarinic receptors of the heart and brain than imperialine (1). The decrease in activity in 2 showed the importance of the 6-keto functionality in imparting the anticholinergic activity.

Puqiedinone, a novel 5 alpha-cevanine alkaloid from the bulbs of Fritillaria puqiensis, an antitussive traditional Chinese medicine.

A novel 5 alpha-cevanine alkaloid, puqiedinone [1], was isolated from the bulbs of Fritillaria puqiensis, a traditional Chinese medicine used widely for its antitussive and expectorant properties. The structure of 1 was assigned as (20R,22S,25R)-20-deoxy-5 alpha-cevanine-3 beta-ol-6-one based on spectral analysis and comparison with the structures of related known compounds. Puqietinone [2] was also identified from the same herbal plant and the structure was revised as (22R,25S)-N-methyl-22,26-epiminocholest-3 beta-ol-6-one based on spectroscopic and X-ray crystallographic methods.