An integrated strategy for characterization of chemical constituents in Stephania tetrandra using LC-QTOF-MS/MS and the target isolation of two new biflavonoids.

LC-MS has been a widely used analytical technique for identification of natural compounds. However, sophisticated and laborious data analysis is required to identify chemical components, especially new compounds, from a large LC-MS dataset. The aim of this study is to develop an integrated data-mining strategy that combines molecular networking (MN), in-house polygonal mass defect filtering (MDF), and diagnostic fragment ion filtering (DFIF) to identify phytochemicals in Stephania tetrandra based on LC-MS data. S. tetrandra samples were prepared by matrix solid-phase dispersion extraction methods and then raw MS spectra were acquired using LC-QTOF-MS/MS. MN and in-house polygonal MDF classified the compounds roughly. Modified DFIF were then used in succession to place each spectrum into a specific class. Finally, the exact structures were deduced by fragmentation pathways and related botanical biogenesis, with the help of the narrowed classification from MN and MDF. The total workflow was a combination of data filtering and identification methods for rapid characterization of known compounds (dereplication) and discovery of new compounds. Consequently, 144 compounds were identified or tentatively identified in the aerial parts and roots of S. tetrandra, including 11 potentially new compounds and 63 compounds first identified in this species. Among 144 compounds, 61 were from the aerial parts exclusively, 8 were from the roots exclusively, and 75 were found in both parts. Furthermore, two new biflavonoids were isolated with the guide of LC-MS analysis and structurally elucidated by spectroscopic methods. In conclusion, the proposed data-mining strategy based on LC-MS can be used to profile chemical constituents with high efficiency and guide the isolation of new compounds from medicinal plants. The comparison of the components of the aerial parts and roots of S. tetrandra would be helpful for the rational utilization of the medicinal plant.

Deep Eutectic Solvent-Based Microwave-Assisted Extraction for the Chromatographic Analysis of Bioactive Flavonoids in Spirodela polyrrhiza.

Deep eutectic solvents (DESs) are regarded as promising solvents to extract chemicals from plant materials. In this study, a DES-based microwave-assisted extraction (MAE) method was developed for the chromatographic analysis of four bioactive flavonoids in Spirodela polyrrhiza. Chromatographic separation was achieved on a Promosil C18-column. Prior to the HPLC analysis, the flavonoids were rapidly extracted by a DES-MAE process using choline chloride/levulinic acid (1:2, mol/mol) as the solvent. The extraction parameters were optimized using response surface methodology, and the optimal DES-MAE was fast and efficient compared with conventional solvent-based MAE and ultrasonic-assisted extraction using DES. The recoveries of optimized DES-MAE for the four flavonoids ranged from 97.80 to 103.29%. This study demonstrates that the validated DES-MAE-HPLC method is efficient, accurate and applicable to the determination of flavonoids in S. polyrrhiza.

Solubilization of Hexyl Aminolevulinate by Surfactants for Tumor Fluorescence Detection.

Hexyl aminolevulinate (HAL) is a lipophilic derivative of 5-aminolevulinic acid (5-ALA) and can induce more protoporphyrin IX (PpIX) formation and stronger fluorescence intensity (FI) than 5-ALA, which will greatly facilitate photodynamic diagnosis and therapy. The main drawback of HAL is its low solubility in neutral aqueous media. In this study, surfactants were used to increase HAL solubility in the cell culture medium and serum, followed by in vitro fluorescence formation measurement in human pancreatic cancer cells (SW1990) and in vivo fluorescence detection in tumor-bearing mice. The results showed that Tween 80 (TW80) and Kolliphor® HS 15 (HS15) increased the solubility of HAL in the selected media. Although TW80 and HS15 exhibited in vitro cytotoxicity at high concentrations (5 mg mL-1 ), they facilitated fluorescent signal formation at the early stage of cell incubation. When surfactants were used, the FI should be determined without the routine washing process because surfactant-containing culture medium caused the loss of synthesized PpIX during the washing process. When HAL dissolved in TW80 solution was injected intraperitoneally into pancreatic cancer-bearing mice at a dose of 50 mg kg-1 , the tumors exhibited red fluorescence, which indicated that systemic administration of surfactant-solubilized HAL might be applicable for tumor fluorescence detection in vivo.

DNA barcodes for the identification of Stephania (Menispermaceae) species.

Stephania is a medicinal plants-rich genus of Menispermaceae. However, the identification of morphologically-similar species in Stephania is difficult using the currently reported methods. The indiscriminate overexploitation of Stephania plants has resulted in clinical misuse and endangerment of many species, which necessitates the development of an efficient and reliable method for species authentication. Therefore, six candidate DNA barcode sequences (ITS, ITS2, psbA-trnH, matK, rbcL, and trnL-F) were tested for their capacity to identify Stephania species. The barcodes were analyzed either as a single region or in combination by tree-based [neighbor-joining (NJ) and Bayesian inference (BI)], distance-based (PWG-distance), and sequence similarity-based (TaxonDNA) methods. Amplification and sequencing success rates were 100% for all six candidate barcodes. A comparison of six barcode regions showed that ITS exhibited the highest number of variable and informative sites (182/179), followed by psbA-trnH (173/162). DNA barcoding gap assessment showed that interspecific distances of the six barcodes were greater than intraspecific distances. The identification results showed that species discrimination rates of combination barcodes were higher than those of single-region barcodes. Based on best match and best close match methods, the ITS+psbA-trnH combination exhibited the highest discrimination power (93.93%). Further, all Stephania species could be resolved in the phylogenetic trees based on ITS+psbA-trnH (NJ, BI). This study demonstrates that DNA barcoding is an efficient method to identify Stephania species and recommends that the ITS+psbA-trnH combination is the best DNA barcode for the identification of Stephania species.

Dextran-based Nanomicelle System with Directly Ester-bound Gadolinium Chelates as a Magnetic Resonance Imaging Contrast Agent for Tumor Detection.

There is a strong need to develop MRI contrast agents (CAs) with lower in-vivo retention, stronger signal enhancement, and more specific imaging. Here, we report a novel dextran (DEX)-based nanomicelle system as an MRI CA with superior tumor imaging and relatively short intravascular persistence. Gadolinium (Gd)-chelate (DTPA-Gd) was conjugated directly to DEX hydroxyl via a degradable ester bond. DEX-DTPA-Gd was then modified with dodecylsuccinic anhydride to obtain the amphiphilic derivative, 2-dodecylsuccinic acid (DSA)-grafted DEX-DTPA-Gd. Nanomicelles were prepared by dissolving DSA-DEX-DTPA-Gd in water using ultrasonication. The physicochemical properties, cytotoxicity, and MRI efficiency of the synthesized CA were evaluated. The synthesized DSA-DEX-DTPA-Gd self-assembled into nanomicelles with an average diameter of 67.80 ± 5.21 nm. Within the given Gd concentration range, DSA-DEX-DTPA-Gd and Magnevist® exhibited similar cytotoxicity. DEX-based CAs resulted in a greater contrast enhancement of T1-weighted signal intensity in the tumor region than Magnevist®, and the tumors were clearly defined for at least 3 h. Simultaneously, the ester bond in DSA-DEX-DTPA-Gd facilitated the elimination of Gd chelates, compared with the relatively more stable amide linker. The DEX-based nanomicelle system with directly ester-bound DTPA-Gd may serve as an MRI CA with superior tumor imaging and relatively rapid elimination.

Determination of caffeoylquinic acid derivatives in Azolla imbricata by chitosan-based matrix solid-phase dispersion coupled with HPLC-PDA.

Five main components of Azolla imbricata were isolated and identified as caffeoylquinic acid derivatives, four of which were isolated from this species for the first time. The five compounds exhibited strong antioxidant activities and were used as chemical markers for quantitative analysis. A chitosan-based matrix solid-phase dispersion (MSPD) extraction coupled with high-performance liquid chromatography (HPLC) was developed for the determination of the five analytes in A. imbricata. The optimal conditions for the chitosan-based MSPD extraction were as follows: low viscosity chitosan HL-1 as the dispersant, sample-to-dispersant mass ratio of 1:1, 10 mL of a methanol-sulfuric acid aqueous solution (0.2 M) (7:3, v/v) as the elution solvent, and a grinding time of 1 min. Compared with ultrasonic assisted extraction (UAE), the chitosan-based MSPD extraction exhibited higher extraction efficiency, consumed less solvent and time, and provided a cleaner extract. Compared with C18-based MSPD, the developed method was less expensive and more environmentally-friendly. The validated MSPD-HPLC method was efficient, reliable, and applicable to the quantification of caffeoylquinic acids in A. imbricata.

Stearic Acid-Grafted Chitooligosaccharide Nanomicelle System with Biocleavable Gadolinium Chelates as a Multifunctional Agent for Tumor Imaging and Drug Delivery.

PURPOSE: Theranostic nanoplatforms are promising approaches for diagnosis and treatment. Here, we report a drug-loaded nanomicelle system with biocleavable gadolinium (Gd) chelates as a multifunctional biodegradable agent for simultaneous magnetic resonance imaging (MRI) and drug delivery. METHODS: Self-assembled nanomicelles based on stearic acid-grafted chitooligosaccharide were utilized as vehicles. Gd chelates, DTPA-Gds, were linked to the nanomicelles via redox-responsive disulfide bonds, and hydrophobic drugs were encapsulated in the micelle cores. MRI and cargo delivery were investigated in orthotopic pancreatic tumor-bearing mice. RESULTS: In vivo MRI demonstrated that the biodegradable agent was cleaved by endogenous thiols after intravenous injection, and the released DTPA-Gds were eliminated rapidly. At the same time, the agent resulted in a greater contrast enhancement of T1-weighted MR signal intensity at the tumor region than Magnevist®, and the tumor boundaries were clearly defined for at least 2 h. In addition, the agent possessed high drug-loading and tumor-targeting capacities. Loading content and encapsulation efficiency of docetaxel were 3.2% and 99.4%, respectively. Compared with Taxotere®, the commercially available docetaxel injection, the docetaxel-loaded agent significantly increased the drug concentration in tumor tissue in vivo. CONCLUSION: The fabricated multifunctional agent may serve as a biodegradable nanoscale MRI contrast agent and as a drug delivery system for tumor diagnosis and treatment.

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 ß1 (TGF-ß1) and interleukin-1ß (IL-1ß) 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-ß1 and IL-1ß. These data indicate the potency of ISOF in treating asthma and also provide insights into the development of new anti-asthma agent.

Formulation and evaluation of a montelukast sodium orally disintegrating tablet with a similar dissolution profile as the marketed product.

A major challenge of orally disintegrating tablet (ODT) development is predicting its bioequivalence to its corresponding marketed product. Therefore, comparing ODT dissolution profiles to those of the corresponding marketed product is very important. The objective of this study was to develop a 5.2-mg montelukast sodium (MS) ODT with a similar dissolution profile to that of the marketed chewable tablet. Dissolution profiles were examined in different media to screen each formulation. We found that MS dissolution from ODTs in acidic medium heavily depended on manufacturing methods. All MS ODTs prepared using direct compression rapidly disintegrated in acidic medium. However, dispersed MS powders aggregated into sticky masses, resulting in slow dissolution. In contrast, MS ODTs prepared using wet granulation had much faster dissolution rates in acidic medium with no obvious aggregation. Additionally, the optimized formulation, prepared using wet granulation, displayed similar dissolution profiles to the marketed reference in all four types of media examined (f2 > 50). The in vitro disintegration time of the optimized ODT was 9.5 ± 2.4 s, which meets FDA requirements. In conclusion, the wet granulation preparation method of MS ODTs resulted in a product with equivalent dissolution profiles as those of the marketed product.

Pharmacokinetics of isoforskolin after administration via different routes in guinea pigs.

1. The objective of this study was to characterize the pharmacokinetics of isoforskolin after oral, intraperitoneal and intravenous administration, as well as to compare bioavailability. 2. Isoforskolin was administered to guinea pigs at a dose of 2 mg/kg. Plasma concentrations were determined by high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) method. The pharmacokinetic parameters were calculated by a noncompartmental method. A compartment model was also adopted to describe the pharmacokinetic profiles. 3. The pharmacokinetic behavior of intravenously administered isoforskolin was characterized by rapid and extensive distribution (Vz = 16.82 ± 8.42 L/kg) followed by rapid elimination from the body (Cl = 9.63 ± 4.21 L/kg/h). After intraperitoneal administration, isoforskolin was absorbed rapidly (Tmax = 0.12 ± 0.05 h). The pharmacokinetic profiles of isoforskolin were similar after intraperitoneal and intravenous administration, except for the concentrations at the initial sampling times. Isoforskolin was also absorbed rapidly following oral dosing; however, the concentration-time data were best fit to a one-compartment model, which was different from that observed after intravenous and intraperitoneal administration. Following intraperitoneal and oral administration, the absolute bioavailability of isoforskolin was 64.12% and 49.25%, respectively. 4. Isoforskolin is a good candidate for oral administration because of its good oral bioavailability.

Single- and multiple-dose pharmacokinetics of a novel tetramethylpyrazine reservoir-type transdermal patch versus tetramethylpyrazine phosphate oral tablets in healthy normal volunteers, and in vitro/in vivo correlation.

A novel reservoir-type transdermal system of 2,3,5,6-tetramethylpyrazine (TMP) was developed containing eucalyptus oil as a penetration enhancer. The single and multiple-dose pharmacokinetic profiles of TMP administrated by TMP transdermal patch were characterized in healthy volunteers using an in vivo, randomized, open-label, two-way crossover design. 2,3,5,6-Tetramethylpyrazine phosphate (TMPP) oral tablets were chosen as reference. Following single/multiple oral administration of 200/100 mg TMPP tablets, a TMP C(max) of 1284/613.5 ng/mL was observed within 0.75 h. Single/multiple applications of the TMP patch yielded mean C(max) of 309/325 ng/mL at a median T(max) of 5/4 h, with steady state achieved at second application. The mean C(min) of the patch was 131±30.38 ng/mL, contrasting to nearly zero for the tablet. Multiple applications of patch produced an accumulative effect over single application. At steady state 250 mg/20 cm(2) TMP patch given daily provided comparable exposure to 100 mg TMPP tablets three times daily (3753.91 versus 3563.67 ng·h/mL). TMP tablets and patch yielded similar steady-state plasma concentrations: C(av) (148.48±51.27, 156.41±40.31 ng/mL). The results demonstrated that TMP patch can achieve a therapeutic effect that is comparable to oral administration, exhibited prolonged and sustained plasma levels, fewer drug fluctuations, lower adverse effects, more convenience, and improved patient compliance. In-vitro permeation through human skin demonstrated zero-order kinetics with the flux of 364 µg/cm(2)/h. The predicted C(av) (163.9 ng/mL) was in agreement with the observed C(av) (156.4 ng/mL).

Development of a reservoir-type transdermal delivery system containing eucalyptus oil for tetramethylpyrazine.

The purpose of this study was to formulate a reservoir-type transdermal delivery system (TDS) for 2,3,5,6-tetramethylpyrazine (TMP) to enable the delivery of a sufficient dose through human skin to achieve an effective therapeutic plasma concentration. To improve the penetration of TMP in the reservoir-type TDS, several chemical penetration enhancers were investigated using in vitro rat dorsal skin permeation studies. Eucalyptus oil was found to enhance the permeation of TMP to the greatest extent, with the optimal concentration being 5% and the flux being 542.6 ± 49.7 µg/cm(2)/h, which was 4.5-fold greater than control with no enhancer (p < 0.01). The flux of the optimized reservoir-type TDS permeated through the human epidermis was 346.0 ± 27.7 µg/cm(2)/h. Based on the in vitro human skin permeation flux and the pharmacokinetics parameters observed, the clinical surface area of the TDS patch was predicted to be 20 cm(2). The in vivo study conducted in rabbits showed that the TMP TDS patch containing 5% eucalyptus oil had a more favorable pharmacokinetic profile, with a lower C(max) and prolonged T(max) and mean residence time than that observed with the oral administration of TMP. The TMP reservoir-type TDS was shown to be a promising alternative route to oral administration or intravenous infusion of TMP.

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.

Determination of palmatine in canine plasma by liquid chromatography-tandem mass spectrometry with solid-phase extraction.

A sensitive and specific high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS-MS) method has been developed and validated for the determination of palmatine in canine plasma. Palmatine and jatrorrhizine (internal standard, I.S.) were extracted from plasma samples by solid-phase extraction (SPE) using Oasis HLB cartridges. The chromatographic separation was performed on a Waters XTerra MS C(18) reversed-phase column at 30 degrees C. The gradient mobile phase, delivered at 0.25 mL/min, was composed of a mixture of acetonitrile -0.1% (v/v) acetic acid aqueous solution adjusted to pH 2.8 with triethylamine. Positive electrospray ionization was utilized as the ionization source. Palmatine and the internal standard (I.S.) were determined using multiple reaction monitoring (MRM) of precursor-->product ion transitions at m/z 352-->336 and m/z 338-->322, respectively. The lower limit of quantification (LLOQ) was 0.1 ng/mL using 100 microL plasma samples and the linear calibration range was from 0.1 to 500 ng/mL. The inter-day and intra-day RSDs were lower than 9.9% and the recoveries of palmatine ranged from 87.3 to 100.9%. The mean extraction recoveries of palmatine and the I.S. were 99.2 and 96.8%, respectively. The method has been successfully applied to the pharmacokinetic studies of palmatine in beagle dogs after oral administration and intramuscular injection of palmatine.

Determination of aglycone conjugated metabolites of scutellarin in rat plasma by HPLC.

A specific, precise and accurate high-performance liquid chromatography (HPLC) method for the determination of aglycone conjugated metabolites of scutellarin in plasma after enzymolysis to scutellarein (the aglycone of scutellarin) was developed and validated. The chromatographic separation was performed on a Lunar C18(2) reversed-phase column at a column temperature of 40 degrees C. The mobile phase, delivered at 1.0 ml/min, consisted of acetonitrile-KH2PO4 buffer (40 mM, pH 2.5) (33:67, v/v). The detection wavelength was set at 335 nm. Scutellarein and I.S. (quercetin) were isolated by a liquid-liquid extraction after incubating the plasma samples with beta-glucuronidase/sulfatase. The method was validated using scutellarin spiked plasma as standards. Linearity was confirmed in the concentration range of 0.2165-4.329 nmol/ml, R.S.D.s were within 8.32%, and the recoveries of scutellarein ranged from 101.2 to 108.6%. The method is applicable to the pharmacokinetic study of aglycone conjugated metabolites of scutellarin in rats after oral administration of scutellarin.

[Determination of ligustrazine in human plasma by high performance liquid chromatography with solid-phase extraction].

A high performance liquid chromatographic method was developed for the determination of ligustrazine in human plasma. The chromatographic separation was performed on a Luna C18 column (150 mm x 4.6 mm i.d., 5 microm) at column temperature of 40 degrees C. The mobile phase, a mixture of methanol-acetonitrile-acetate buffer of pH 5.0 (50:8:42, v/v), was delivered at a flow rate of 1.0 mL/min. The detection wavelength was 280 nm. Plasma samples were prepared with a C8 solid-phase extraction column. Linearity was confirmed in the mass concentration range of 25-5000 microg/L with the correlation coefficient of 0.9999. The extraction recovery of ligustrazine ranged from 96.72% to 100.90%. The relative standard deviations (RSDs) of intra- and inter-day assay at the mass concentrations of 50, 500 and 3000 microg/L were less than 8.64% and the accuracies were between 99.59%-103.26%. The limit of detection (LOD) was 10 microg/L. The results of this method validation satisfactorily meet the acceptance criteria of bioanalysis and the method is applicable to the pharmacokinetic studies of ligustrazine in human beings.

A pharmacokinetic study of intramuscular administration of bulleyaconitine A in healthy volunteers.

The pharmacokinetics of bulleyaconitine A (BLA) after a single dose of 0.2 mg intramuscular injection was evaluated in healthy volunteers. Physical exam, vital signs, clinical laboratory tests and electrocardiogram measurements were monitored to assess the safety and tolerance of the drug. The plasma levels of BLA in serial samples, collected over 15 h, were measured by a validated high-performance liquid chromatography (HPLC)-electrospray ionization tandem mass spectrometry (MS-MS) method. It was demonstrated that BLA was absorbed rapidly after intramuscular injection. The pharmacokinetic parameters were as follows: the t(max) value was 0.90+/-0.68 h, the C(max) value was 1.13+/-0.76 ng/ml, the AUC(0-t) was 5.16+/-2.05 ng.h/ml, and t(1/2) was found to be 4.88+/-0.97 h. No subject showed any drug-related clinically significant changes on physical examination, vital signs or laboratory tests. Eight of ten subjects reported a distinct feeling of pain at the site of injection starting approximately at the time of their peak plasma concentration and lasting for 2-6 h. The pain was tolerable, and no subject required additional treatment.

Determination of bulleyaconitine A in human plasma by liquid chromatography-electrospray ionization tandem mass spectrometry.

A sensitive and specific high-performance liquid chromatography (HPLC)-electrospray ionization tandem mass spectrometry (MS-MS) was developed for the determination of bulleyaconitine A (BLA) in human plasma. BLA and internal standard (I.S.) ketoconazole were extracted from the plasma by a liquid-liquid extraction. The supernatant was evaporated to complete dryness and reconstituted with acetonitrile containing 0.1% acetic acid before injecting into an ODS MS column. The gradient mobile phase was composed of a mixture of acetonitrile (containing 0.1% acetic acid, v/v) and 0.1% acetic acid aqueous solution eluted at 0.3 ml/min. BLA and I.S. were determined by multiple reaction monitoring using precursor-->product ion combinations at m/z 644.6-->584.3 and 531.2-->81.6, respectively. Linearity was established for the concentration range of 0.12-6 ng/ml. The recoveries of BLA ranged from 96.93 to 113.9% and the R.S.D. was within 20%. The method is rapid and applicable to the pharmacokinetic studies of BLA in human.