Mechanistic and therapeutic study of novel anti-tumor function of natural compound imperialine for treating non-small cell lung cancer.

ETHNOPHARMACOLOGICAL RELEVANCE: Bulbus Fritillaria cirrhosa D. Don (BFC) is a Chinese traditional herbal medicine that has long been used as an indispensable component in herbal prescriptions for bronchopulmonary diseases due to its well-established strong anti-inflammation and pulmonary harmonizing effects. Interestingly, there are few case reports in traditional Chinese medicine available where they found it to contribute in anti-tumor therapies. Imperialine is one of the most favored active substances extracted from BFC and has been widely recognized as an anti-inflammatory agent. AIM OF THE STUDY: The aim of the current work is to provide first-hand evidences both in vitro and in vivo showing that imperialine exerts anti-cancer effects against non-small cell lung cancer (NSCLC), and to explore the molecular mechanism of this anti-tumor activity. It is also necessary to examine its systemic toxicity, and to investigate how to develop strategies for feasible clinical translation of imperialine. MATERIALS AND METHODS: To investigate anti-NSCLC efficacy of imperialine using both in vitro and in vivo methods where A549 cell line were chosen as in vitro model NSCLC cells and A549 tumor-bearing mouse model was constructed for in vivo study. The detailed underlying anti-cancer mechanism has been systematically explored for the first time through a comprehensive set of molecular biology methods mainly including immunohistochemistry, western blot and enzyme-linked immunosorbent assays. The toxicity profile of imperialine treatments were evaluated using healthy nude mice by examining hemogram and histopathology. An imperialine-loaded liposomal drug delivery system was developed using thin film hydration method to evaluate target specific delivery. RESULTS: The results showed that imperialine could suppress both NSCLC tumor and associated inflammation through an inflammation-cancer feedback loop in which NF-κB activity was dramatically inhibited by imperialine. The NSCLC-targeting liposomal system was successfully developed for targeted drug delivery. The developed platform could favorably enhance imperialine cellular uptake and in vivo accumulation at tumor sites, thus improving overall anti-tumor effect. The toxicity assays revealed imperialine treatments did not significantly disturb blood cell counts in mice or exert any significant damage to the main organs. CONCLUSIONS: Imperialine exerts anti-cancer effects against NSCLC both in vitro and in vivo, and this previously unknown function is related to NF-κB centered inflammation-cancer feedback loop. Imperialine mediated anti-cancer activity is not through cytotoxicity and exhibit robust systemic safety. Furthermore, the liposome-based system we commenced would dramatically enhance therapeutic effects of imperialine while exhibiting extremely low side effects both on cellular and in NSCLC model. This work has identified imperialine as a promising novel anti-cancer compound and offered an efficient target-delivery solution that greatly facilitate practical use of imperialine.

Isosteroid alkaloids with different chemical structures from Fritillariae cirrhosae bulbus alleviate LPS-induced inflammatory response in RAW 264.7 cells by MAPK signaling pathway.

Isosteroid alkaloids, natural products from Fritillariae Cirrhosae Bulbus, are well known for its antitussive, expectorant, anti-asthmatic and anti-inflammatory properties. However, the anti-inflammatory effect and its mechanism have not been fully explored. In this study, the anti-inflammatory activitives and the potential mechanisms of five isosteroid alkaloids from F. Cirrhosae Bulbus were investigated in lipopolysaccharide (LPS)-induced RAW264.7 macrophage cells. The pro-inflammatory mediators and cytokines were measured by Griess reagent, ELISA and qRT-PCR. The expression of MAPKs was investigated by western blotting. Treatment with the five isosteroid alkaloids in appropriate concentrations could reduce the production of nitric oxide (NO), tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) in supernatant, and suppressed the mRNA expressions of TNF-α and IL-6. Meanwhile, the five isosteroid alkaloids significantly inhibited the phosphorylated activation of mitogen activated protein kinase (MAPK) signaling pathways, including extracellular signal-regulated kinase (ERK1/2), p38 MAPK and c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK). These results demonstrated that isosteroid alkaloids from F. Cirrhosae Bulbus exert anti-inflammatory effects by down-regulating the level of inflammatory mediators via mediation of MAPK phosphorylation in LPS-induced RAW264.7 macrophages, thus could be candidates for the prevention and treatment of inflammatory diseases.

Pharmacokinetics, tissue distribution and excretion of verticinone from F. hupehensis in rats.

Verticinone, the main active component in F. hupehensis, exhibits potent antitussive and expectorant effects. Here, a LC-MS method was developed and applied to study the pharmacokinetics, tissue distribution and excretion of verticinone in rats, and its plasma protein binding in vitro. A significant gender difference in the pharmacokinetics of verticinone in rats was observed, as its absolute oral bioavailability in male and female rats was 45.8% and 2.74%, respectively. The relative bioavailability of verticinone was significantly lower in female rats as compared to male, following intragastrical (i.g.) and intravenous (i.v.) administration. After successive i.g. administration of verticinone, accumulation was observed in female rats but not in the male ones. The tissue distribution study showed that verticinone had a good tissue penetrability and a high tissue affinity in most studied tissues, except brain. After a 2 mg/kg oral dose, less than 4% of the dose was excreted as unchanged parent compound in male rats, and less than 1% in female rats, which indicated that verticinone was metabolized more extensively in female rats than in male rats.

[Rat intestinal absorption trait of peimine and peiminine in Thunberg fritillary bulb extract].

To study the in situ intestinal absorption kinetics and compatibility influence of peimine and peiminine in rats, the absorption of peimine and peiminine in small intestine (duodenum, jejunum and ileum) and colon of rats was investigated using in situ single-pass perfusion method and the drug content was measured by HPLC-ELSD. Perfusion rate, pH, concentration of drug, gender and bile duct ligation can significantly affect the absorption of peimine and peiminine, the Ka, and Papp values in the condition of pH 6.8 and pH 7.4 had significant difference (P<0.01), as drug concentration irlcreased, the absorption parameters of peimine and peiminine decreased, Ka and Papp between low concentrations and middle concentrations was significant difference (P<0.01). Verapamil can not affect Ka and Papp of peimine and peiminine which are in the extract (P> 0.05). Bitter almonds and licorice can significantly reduce the absorption of peimine and peiminine with the usual dose (P<0.01), extracted separately and together had no significant difference on Ka and Papp (P> 0.05). Experimental results show that the absorption features of peimine and peiminine are basically the same, both of them could be absorbed at all segments of the intestine in rats and had no special absorption window, and with significant differences between male and female individuals. The absorption of peimine and peiminine complies with the active transport and facilitated diffusion in the general intestinal segments. Bitter almond and licorice can reduce the intestinal absorption rate ofpeimine and peiminine.

[Comparison on pollen morphology among populations of Changium smyrnioides].

OBJECTIVE: To observe the pollen morphological differences among different populations of Changium smyrnioides. METHOD: The pollen morphology of 10 populations were examined through LM and SEM observations. RESULT: Pollens in different populations were distinguished from each other in the size, the largest average size was the pollen of the population cultivated in Hongshan, and the smallest was that of the population cultivated in Jiuhuashan. Pollens were oval-shaped in all of the populations, and P/E values were around 1.5. Typical feature of surface ornamentation was stripe-like structure, different populations were distinguished from each other in the texture depth and the gap. With different length and width in different populations, typical feature of germinal aperture was nearly square and 3 germinal furrows. Variation with 4 germinal apertures were found in the pollen of population cultivated in Hongshan. CONCLUSION: Diversity of pollen morphology was high, and differentiation was strong in Ch. smyrnioides.

Turbulent-flow chromatography coupled on-line to fast high-performance liquid chromatography and mass spectrometry for simultaneous determination of verticine, verticinone and isoverticine in rat plasma.

A method based on the on-line turbulent-flow chromatography and fast high-performance liquid chromatography/mass spectrometry (TFC-LC/MS) was developed for sensitive and high throughput pharmacokinetic study of traditional Chinese medicines (TCMs). In this method, an on-line extraction column (Waters Oasis HLB) and a fast HPLC column with sub-2 microm particle size (Agilent Zorbax StableBond-C(18), 4.6 mm x 50 mm, 1.8 microm) in a column-switching set-up were utilized. HLB is a reversed-phase extraction column with hydrophilic-lipophilic balanced copolymer (2.1 mm x 20 mm, 25 microm particle size), which will exhibit some turbulent-flow properties at a high-flow rate. The method combines the speed and robustness of turbulent-flow extraction and the sensitivity and separation efficiency of fast HPLC-MS to analyze multiple and trace constituents of TCMs in plasma matrix. This method was successfully applied for pharmacokinetic study of verticine, verticinone and isoverticine, the chemical markers of Fritillaria thunbergii, after oral administration of total steroidal alkaloids extract of F. thunbergii to rats. Each plasma sample was analyzed within 7 min. The method demonstrated good linearity (R>0.999) ranged from 0.505 to 96.0 ng/mL with satisfactory accuracy and precision, and the lower limit of quantifications of verticine, verticinone and isoverticine were estimated to be 0.120, 0.595 and 0.505 ng/mL, respectively. These results indicate that the proposed method is fast, sensitive, and feasible for pharmacokinetic study of TCMs.