The Isosteroid Alkaloid Imperialine from Bulbs of Fritillaria cirrhosa Mitigates Pulmonary Functional and Structural Impairment and Suppresses Inflammatory Response in a COPD-Like Rat Model.

Chronic obstructive pulmonary disease (COPD) is the third leading cause of death in the world. Present therapies for COPD have limited effect on reducing the progression of COPD and suppressing the inflammatory response in the lung. Bulbs of Fritillaria cirrhosa D. Don (BFC) have been used in many Asian countries for a long time to treat pulmonary diseases, such as cough, expectoration, and asthma. Steroidal alkaloids are the major biological active constituents in BFC, whereby imperialine is one of the important steroidal alkaloids. So far, there are no studies reporting the effect of imperialine on COPD. In this study, we investigated the effect of imperialine on pulmonary function and structure and inflammation in a COPD-like rat model which was induced by the combination of exposure to CS and intratracheal administration of LPS. Our data show that imperialine mitigates pulmonary functional and structural impairment and suppressed inflammatory response in a COPD-like rat model by mediating expression of related cytokines in lung tissues of the COPD-like rats, such as IL-1ß, IL-6, IL-8, TNF-α, NF-κB, TGF-ß1, MMP-9, and TIMP-1.

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.

[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.

Stabilizing parallel G-quadruplex DNA by a new class of ligands: two non-planar alkaloids through interaction in lateral grooves.

Human DNA sequences consisting of tandem guanine (G) nucleotides can fold into a four-stranded structure named G-quadruplex via Hoogsteen hydrogen bonding. As the sequences forming G-quadruplex exist in essential regions of eukaryotic chromosomes and are involved in many important biological processes, the study of their biological functions has currently become a hotspot. Compounds selectively binding and stabilizing G-quadruplex structures have the potential to inhibit telomerase activity or alter oncogene expression levels and thus may act as antitumor agents. Most of reported G-quadruplex ligands generally have planar structures which stabilize G-quadruplex by pi-pi stacking. However, based on a pharmacophore-based virtual screening two non-planar G-quadruplex ligands were found. These two ligands exhibit good capability for G-quadruplex stabilization and prefer binding to paralleled G-quadruplex rather than to duplex DNA. The binding of these ligands to G-quadruplex may result from groove binding at a 2:1 stoichiometry. These results have shown that planar structures are not essential for G-quadruplex stabilizers, which may represent a new class of G-quadruplex-targeted agents as potential antitumor drugs.

Sipeimine-producing endophytic fungus isolated from Fritillaria ussuriensis.

Ten strains of endophytic fungi were isolated from the bulbs of the traditional Chinese medicinal plant Fritillaria ussuriensis. The extract from one of them, Fu7, showed a positive reaction with Dragendorff's reagent and the same Rf value in thin-layer chromatography (TLC) analysis as authentic sipeimine. A further TLC scan and high-performance liquid chromatography-evaporative light-scattering detection (HPLC-ELSD) showed that one ingredient of the extract of strain Fu7 had a similar absorption curve in the range 200-700 nm and the same retention time as authentic sipeimine. Thus, the fungus produces the bioactive ingredient sipeimine, as does its host plant, and could be used for the production of sipeimine by fermentation.

Muscarinic M(2) receptors are not primarily involved in the contraction of guinea-pig gallbladder smooth muscle.

The presence of M(1)-M(4) receptors in guinea-pig gallbladder smooth muscle cells has been reported recently. The majority of these receptors are said to be of M(2) subtype. However, there are controversial reports about the functional muscarinic receptors that mediate contraction in this tissue. Similar to gallbladder, it was claimed that M(4) receptors mediate guinea-pig uterine contractions, but these receptors have appeared to be of M(2) subtypes later. Therefore, the antagonistic affinities of three M(2)-selective muscarinic antagonists were determined in contraction and radioligand binding experiments in guinea-pig gallbladder in the present study. The antagonistic affinity values (p K(i)) of gallamine, tripitramine and imperialine were as follows, respectively: 6.28+/-0.15, 8.65+/-0.10 and 6.55+/-0.07 against 0.250 n m [(3)H]QNB binding. All three antagonists displaced the concentration- response curves to carbachol to the right in parallel without affecting the maximum responses. The p A(2) values obtained from constrained Schild plots (-log K(B)) were 4.14+/-0.18 for gallamine, 6.79+/-0.09 for tripitramine, and 7.02+/-0.09 for imperialine. The antagonistic affinity values of gallamine, tripitramine and imperialine for M(2) receptors are reported to be 6. 3, 9.6, 7.7, respectively. The p A(2) values obtained in this study clearly indicate that the primary muscarinic receptors involved in carbachol-induced guinea-pig gallbladder contraction are not of M(2) subtype. The poor correlation between the antagonistic affinity values of these antagonists obtained at radioligand binding (p K(i)) and contraction (p A(2)) experiments also support the conclusion that the majority of muscarinic receptors which have been reported to be of M(2) do not mediate the contractile responses.

Subtypes of muscarinic receptors in rat duodenum: a comparison with rabbit vas deferens, rat atria, guinea-pig ileum and gallbladder by using imperialine.

The specific binding of [3H]QNB to rat duodenum smooth muscle membranes was a saturable process and Scatchard transformation of the saturation curves indicated a linear plot (nH = 1.017+/-0.071). The K(D) and Bmax values were 0.168+/-0.025 nM and 46.7+/-8.6 fmol/mg protein, respectively. Analyses of competition curves using pirenzepine and guanylpirenzepine indicated more than one class of binding site. A minor population of muscarinic binding sites showed high affinity (M1) for both pirenzepine (19.3+/-1.2%; pKi = 8.29+/-0.36) and guanylpirenzepine (29.4+/-2.0%; pKi = 7.28+/-0.11). The antagonistic affinity values of pirenzepine and guanylpirenzepine for the remaining low affinity binding sites, and that of methoctramine indicated the presence of both M2 and M3 subtypes. McN-A-343 produced relaxations in rat duodenum and inhibited twitch contractions of rabbit vas deferens induced by electrical stimulation in a concentration dependent manner. Carbachol (Cch) exerted concentration-dependent negative inotropic effect in rat atria and contractile effects in guinea-pig gallbladder and ileum longitudinal muscle-myenteric plexus preparation. Imperaline displaced the concentration-response curves to McN-A-343 and Cch to the right in parallel, without affecting the maximum responses in all tissues studied. The rank order of the pA2 values was rabbit vas deferens > rat atria > guinea-pig gallbladder = guinea-pig ileum > rat duodenum. The presynaptic muscarinic receptors at the rat duodenum and rabbit vas deferens were concluded to be of M1 and M4 subtypes, respectively.

3-alpha-Chlorimperialine: an M2-selective muscarinic receptor antagonist that penetrates into brain.

Muscarinic M2 receptors have been found to be severely depleted in post-mortem brains of Alzheimer's patients. This loss of receptor may represent a useful diagnostic marker, if it could be quantitatively imaged with single-photon emission computed tomography (SPECT) or positron emission tomography (PET) imaging. In order to develop a radioligand with selectivity for muscarinic M2 receptors, we now report that 3-alpha-chlorimperialine is a potent M2 receptor antagonist with a Ki of 0.32 nM at M2 receptors, a 12-fold selectivity for M2 over M1 receptors, and a 5-fold selectivity for M2 over M4 receptors. Furthermore, 2% of the injected dose of 3-alpha-chlorimperialine per gram tissue penetrates into brain within 30 min, then washes out gradually. Taken together, these studies demonstrate that 3-alpha-chlorimperialine is a potent M2-selective muscarinic antagonist that penetrates into brain and may be a useful substrate for radioiodination and subsequent imaging of brain muscarinic M2 receptors.