Jianming Huang

Intestinal permeability of forskolin by in situ single pass perfusion in rats.

The intestinal permeability of forskolin was investigated using a single pass intestinal perfusion (SPIP) technique in rats. SPIP was performed in different intestinal segments (duodenum, jejunum, ileum, and colon) with three concentrations of forskolin (11.90, 29.75, and 59.90 µg/mL). The investigations of adsorption and stability were performed to ensure that the disappearance of forskolin from the perfusate was due to intestinal absorption. The results of the SPIP study indicated that forskolin could be absorbed in all segments of the intestine. The effective permeability (P (eff)) of forskolin was in the range of drugs with high intestinal permeability. The P (eff) was highest in the duodenum as compared to other intestinal segments. The decreases of P (eff) in the duodenum and ileum at the highest forskolin concentration suggested a saturable transport process. The addition of verapamil, a P-glycoprotein inhibitor, significantly enhanced the permeability of forskolin across the rat jejunum. The absorbed fraction of dissolved forskolin after oral administration in humans was estimated to be 100 % calculated from rat P (eff). In conclusion, dissolved forskolin can be absorbed readily in the intestine. The low aqueous solubility of forskolin might be a crucial factor for its poor oral bioavailability.

Identification of Alkaloids in Stephania hainanensis by Liquid Chromatography Coupled with Quadrupole Time‐of‐flight Mass Spectrometry

INTRODUCTION Plants in the genus Stephania can produce diverse bioactive alkaloids. Stephania hainanensis is a medicinal plant that contains effective alkaloids. However, only 10 alkaloids have been reported in this species. OBJECTIVE To characterise the alkaloids in Stephania hainanensis using liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (LC-QTOF-MS/MS). METHODS An LC-QTOF-MS/MS method was developed for structural characterisation of the alkaloids in Stephania hainanensis. The chromatographic separation was performed on a phenyl column with gradient elution, and the tandem mass spectra were obtained by using an electrospray ionisation (ESI) interface in positive ionisation mode. Compound identification was based on the exact masses, fragmentation pathways, retention behaviours and related botanical biogenesis. RESULTS A total of 37 tetrahydroprotoberberine-, quaternary protoberberine-, aporphine-, proaporphine-, benzylisoquinoline- or bisbenzylisoquinoline-type alkaloids were identified or tentatively identified in a single LC run. Twenty-seven of these alkaloids, including the benzylisoquinoline-type of alkaloids, have not been previously reported in Stephania hainanensis. The possible fragmentation pathways of different types of alkaloids were proposed. Besides the general fragmentations, the characteristic losses of CH3 N = CH2 were observed for the benzylisoquinoline and aporphine alkaloids with two methyl groups on the nitrogen. CONCLUSION The LC-QTOF-MS/MS method enabled profiling and rational, but tentative, identification of diverse alkaloids in Stephania hainanensis. The results obtained may be helpful for understanding the bioactivity of S. hainanensis and evaluating the quality of this plant. Copyright

Matrix Solid‐phase Dispersion Extraction for Chromatographic Analysis of Labdane Diterpenoids in Coleus forskohlii

INTRODUCTION The quality of Coleus forskohlii is often evaluated by high performance liquid chromatography (HPLC), using bioactive labdane diterpenoids as chemical markers. However, the existing sample preparation methods for the analysis of diterpenoids in C. forskohlii are generally labour-intensive, time-consuming and require large volumes of solvents. OBJECTIVE To establish an efficient matrix solid-phase dispersion (MSPD) extraction method for the simultaneous analysis of five bioactive diterpenoids in C. forskohlii by HPLC. METHODOLOGY Herbal samples were prepared by an optimised MSPD procedure using C(18) as the sorbent. The quantification of the diterpenoids was achieved by HPLC with evaporative light scattering detector (ELSD), and the identification of the five compounds was performed by HPLC with tandem mass detector (MS/MS). The efficiency of the MSPD method was also compared with other extraction techniques including Soxhlet extraction, heat reflux extraction, ultrasonic-assisted extraction and microwave-assisted extraction. RESULTS The MSPD extracted five diterpenoids with satisfactory recoveries ranging from 98.36% to 102.08%. Compared with other extraction methods, the proposed MSPD method had the advantages of combining extraction and clean-up into a single step, consuming less time and requiring lower solvent volumes. CONCLUSION The MSPD method is simple, rapid and efficient for the extraction of labdane diterpenoids from C. forskohlii. The MSPD procedure coupled with HPLC-ELSD or HPLC-MS/MS is suitable for the quantification and identification of the diterpenoids in C. forskohlii.

A sensitive and specific HPLC-MS/MS analysis and preliminary pharmacokinetic characterization of isoforskolin in beagle dogs.

A sensitive and specific high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) method has been developed and validated for the determination of isoforskolin in canine plasma. Liquid-liquid extraction was used to extract isoforskolin and the internal standard (I.S.) eplerenone from canine plasma. The chromatographic separation was performed on an Agela Venusil XBP Phenyl column with an isocratic mobile phase consisting of methanol-2mM ammonium acetate-formic acid (62:38:0.1, v/v/v), pumped at 0.35 mL/min. Isoforskolin and I.S. were detected at m/z 433.4→373.3 and m/z 415.3→163.5 in positive ion and multiple reaction monitoring (MRM) mode, respectively. The standard curves were linear over the concentration range of 0.1-200 ng/mL (r>0.99). The intra- and inter-batch accuracy values for isoforskolin at four concentrations were 90.2-108.3% and 97.8-106.6%, respectively. The RSDs were less than 6.0%. The mean extraction recoveries of isoforskolin and I.S. were 97.0 and 88.4%, respectively. The method was successfully applied to the pharmacokinetic study after an intravenous administration of isoforskolin in beagle dogs.

Molecular phylogeny of Chinese Stephania (Menispermaceae) and reassessment of the subgeneric and sectional classifications

Abstract. Many species of Stephania Lour. are used traditionally in South-east Asia as medicinal plants. Understanding and predicting their therapeutic properties could be improved, provided that the evolutionary relationships among lineages are clarified. We present the first molecular phylogeny of the genus Stephania, focusing on the species occurring in China on the basis of nuclear (internal transcribed spacer, ITS) and chloroplast (trnL–F) markers sequenced from 29 species of Stephania. Our results showed that S. subgenus Stephania and S. subgenus Tuberiphania are not monophyletic, owing to the phylogenetic placement of a single species (S. mashanica). The relationships with the third subgenus, S. subgenus Botryodiscia, are not resolved. None of the sections in our analyses is monophyletic. Our study calls for further phylogenetic investigations including more accessions from the whole distribution area of the genus. A taxonomic revision of the genus Stephania, which would reassess the appropriateness of the macromorphological characters used so far to distinguish among subgenera (e.g. flower merism, size and aspect of the rootstock and main root), and sections (e.g. inflorescence morphology, sessiliflorous or not), is much needed.

The predictive value of the prealbumin-to-fibrinogen ratio in patients with acute pancreatitis

Early identification of severe acute pancreatitis (SAP) progression is important in acute pancreatitis (AP) treatment. The Ranson, APACHE II and CTSI systems are difficult to use and exhibit limited predictive value. Prealbumin and fibrinogen are acute phase reactants generally used to assess the nutritional statuses and coagulation functions of AP patients, respectively. Here, we explored the value of the combination of these two markers for evaluating AP severity and prognosis.

Inhibition of airway remodeling and inflammation by isoforskolin in PDGF-induced rat ASMCs and OVA-induced rat asthma model

Isoforskolin (ISOF) has been reported to play an important role in many illnesses including respiratory, cardiovascular and ophthalmologic diseases. In our study, we aimed to investigate how ISOF regulates airway remodeling and inflammation in asthma. Based on SO2-stimulated mouse cough model, we assessed the role of ISOF in cough and secretion of phlegm. Afterwards, platelet derived growth factor (PDGF)-induced primary rat airway smooth muscle cell (ASMC) model and ovalbumin (OVA)-induced rat asthma model were used to continue our following research. Our results showed that ISOF could prolong the cough latent period, reduce the cough times in two minutes, and increase the excretion of red phenol, which suggested the antitussive and expectorant effects of ISOF. Besides, ISOF pretreatment reversed the hypotonicity and cytoskeleton remodeling in PDGF-induced ASMCs, and reduced mucus hypersecretion and collagen overdeposition in OVA-induced rat asthma model, which indicated its inhibition on airway remodeling in vitro and in vivo. Moreover, ISOF reduced the invasion of inflammatory cells into bronchoalveolar lavage fluid (BALF) and lungs, which revealed its inhibitory role in airway inflammation. The down-regulation of transforming growth factor β1 (TGF-β1) and interleukin-1β (IL-1β) upon ISOF treatment might be responsible for its anti-remodeling and anti-inflammation roles. In conclusion, ISOF can reduce cough and sputum, as well as inhibit airway remodeling and inflammation by regulating the expression of TGF-β1 and IL-1β. These data indicate the potency of ISOF in treating asthma and also provide insights into the development of new anti-asthma agent.

A fast and sensitive HPLC–MS/MS analysis and preliminary pharmacokinetic characterization of cudratricusxanthone B in rats

A method for the quantitative analysis of cudratricusxanthone B (CXB) in rat plasma by high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) has been developed and validated. The method involved liquid-liquid extraction from plasma, simple chromatographic conditions on a Venusil XBP-PH C(18) column with the mobile phase of 0.5% formic acid in methanol, and mass spectrometric detection using an API-3000 instrument. Multiple reaction monitoring (MRM) mode was used to monitor precursor/product ion transitions of m/z 397.1/285.0 for CXB and m/z 381.6/269.2 for the internal standard (I.S.) cudraxanthone H. The standard curves were linear over the concentration range of 1-500 ng/mL for CXB in rat plasma. The intra- and inter-batch accuracy for CXB at four concentrations was 89.4-99.5% and 89.4-100.8%, respectively. The RSDs were less than 7.92%. The lower limit of quantification for CXB was 1.0 ng/mL using 100 microL of plasma. The average extraction recoveries of CXB ranged from 80.1 to 95.4% at the concentrations of 2, 50 and 500 ng/mL, respectively. This method was successfully applied to the pharmacokinetic study after an intravenous administration of CXB in male Sprague-Dawley (SD) rats.

Structural Characterisation of Alkaloids in Leaves and Roots of Stephania kwangsiensis by LC‐QTOF‐MS

INTRODUCTION The tuberous roots of Stephania kwangsiensis, which contain bioactive alkaloids, are used as a traditional Chinese medicine. Overexploitation of the roots has made the plant increasingly rare, and the abundant leaves of the same plant may offer a potential alternative. However, there is insufficient phytochemical information for a comparison of alkaloid compositions in the two parts. OBJECTIVE To characterise and compare the alkaloids in the leaves and roots of S. kwangsiensis. METHODS The alkaloids in S. kwangsiensis were characterised using high pressure liquid chromatography coupled with positive electrospray ionisation quadrupole time-of-flight tandem mass spectrometry (HPLC-(+)ESI-QTOF-MS/MS). The alkaloid compositions in the leaves and roots were compared by visual inspection combined with principal component analysis (PCA) of the HPLC-MS data. RESULTS Seventy-five alkaloids comprising aporphine-, proaporphine-, protoberberine-, benzylisoquinoline-, bisbenzylisoquinoline- and morphine-type alkaloids were identified or tentatively identified in the roots and leaves of S. kwangsiensis. Sixty-three of these alkaloids have not been previously reported in this species, and three have not been previously reported in the literature. The roots and leaves had similarities in alkaloid composition but differences in the peak intensities of most alkaloids. The PCA revealed that the samples were clustered into two distinct groups, which corresponded to leaves and roots. CONCLUSION This study further clarified the chemical constituents in the roots of S. kwangsiensis, and revealed that diverse alkaloids were also present in the leaves. The comparative chemical profiling of the two parts provides useful information on their potential medicinal use. Copyright

Leaf epidermal features of Chinese Stephania Lour. (Menispermaceae) and their systematic significance

SummaryA previously published molecular phylogeny of Chinese species of Stephania (Menispermaceae) showed that none of the sections sensu Luo, recognised based on flower, inflorescence, leaf, and tuber traits, was monophyletic. In order to assess the use of an additional vegetative character for supraspecific classification in Stephania, we conducted an analysis of leaf micromorphology. Leaf epidermal features of 34 out of the c. 40 Chinese species of Stephania were investigated using light and scanning electron microscopy. Leaf epidermal characters were found to be constant at species level, but variable among species. The distribution pattern of papillae on the epidermal cells of the adaxial and abaxial sides of the leaves revealed three groups in Stephania, fitting with the recognition of three subgenera. Combined with the latter character, the shape of anticlinal cell walls proved to be useful to discriminate between sections, at least in subgenus Stephania. In addition, our results show that S. chingtungensis (subg. Stephania) exhibits leaf micromorphological states characteristic of subgenus Tuberiphania. The systematic position of the recently described S. novenanthera and of the taxonomically challenging S. excentrica within subgenus Tuberiphania is suggested by leaf epidermal features.