Development and validation of a sensitive liquid chromatography-tandem mass spectrometry method for the assay of 12 substances in rat plasma and its application to rat pharmacokinetics of Epimedium and Psoraleae Fructus herb pair after oral administration.

Epimedium (EM) and Psoraleae Fructus (PF) are a traditional herb combination often used as a fixed form to treat osteoporosis disease in the clinic. However, the intricate interactions of this pair remain unknown. In our study, we undertook a comprehensive examination of their compatibility behaviors. Concurrently, a precise and sensitive quantitation method was successfully developed and validated using liquid chromatography-tandem mass spectrometry for the determination of 12 components. This method was applied in analyzing herbal extracts and biological samples (both in the portal vein and systemic plasma), which was also used to study the pharmacokinetics of the herb pair. The results indicated that the combination of EM and PF enhanced the dissolution of chemical components from PF in extracts, but it had a negligible influence on the contents of the components from EM. On the contrary, the in vivo exposure of the lowly exposed EM flavonoids significantly increased following the combination of EM and PF, whereas the highly exposed psoralen and isopsoralen were greatly reduced. These interactions might be crucial for the synergy and toxicity reduction of the herbal pair in disease treatment, which pave the way for further exploration into the clinical application and pharmacological mechanisms of EM and PF.

Discovery and evaluation of cytisine N-isoflavones as novel EGFR/HER2 dual inhibitors.

Aberrant signaling of EGFR (ErbB) family members, in particular epidermal growth factor receptor (EGFR) and human epidermal growth factor 2 (HER2), is associated with the occurrence and development of many types of human malignancies (e.g., breast, lung, and gastric cancers), and dual targeting of EGFR/HER2 by small-molecular inhibitors has proven to be an effective therapeutic approach for treating these cancers. Herein we extracted and isolated from the medicinal plant Sophora alopecuroides L. a new natural product, dubbed Cytisine N-methylene-(4',7-dihydroxy-3'-methoxy)-isoflavone (CNI1) that features a unique molecular framework. Our biochemical kinase assay suggested that one of its derivative CNI3 exhibited the best, micromolar (µM) inhibition activities against the EGFR (IC50 of 1.1 µM; Ki of 0.6 µM) and HER2 (IC50 of 3.5 µM; Ki of 1.8 µM) kinases. By contrast, another derivative CNI4 was most potent in inhibiting the EGFR-overexpressing A431 cancer cell line (IC50 of 45.5 µM) and the HER2-overexpressing BT-474 cancer cell line (IC50 of 32.9 µM), while the respective cellular activities of Lapatinib (a marketed drug) were 24.9 and 20.3 µM under the same assay condition. Moreover, both CNI3 and CNI4 showed desirable anti-metastatic efficacy in another two breast cancer models (viz., MDA-MB-231 and 4T1). In addition, we explored the inhibitory mechanisms of the CNIs against EGFR and HER2 by molecular dynamics simulation and revealed a novel mode of action that engages the cytisine and chromone moieties in CNIs. By combining structure- and ligand-based analysis, we further rationally engineered a new CNI compound that exhibits considerably improved cytotoxicity against both types of A431 and BT-474 cancer cells. Our study demonstrates the CNI compounds as a new class of EGFR/HER2 dual inhibitors and paves a way for their further development.

Measuring Prospective Imagery: Psychometric Properties of the Chinese Version of the Prospective Imagery Task.

OBJECTIVE: Prospective negative imagery is suggested to play an important role in the development and maintenance of anxiety and depression. The Prospective Imagery Task (PIT) was developed to assess prospective imagery. Given the importance of prospective imagery for mental health in the Chinese cultural context, our objective was to examine the psychometric properties of the PIT in a Chinese sample. METHODS: The instrument was validated among a sample of 1,372 Chinese individuals (mean age = 19.98, SD = 4.57; 35.2% male) who completed the PIT immediately following the Beck Depression Inventory-II (BDI-II) and State-Trait Anxiety Inventory-Trait version (STAI-T). RESULTS: The two-factor structure of the PIT was in line with the original study, with satisfactory reliability and positive correlations with the BDI-II and STAI-T scores. Latent profile analysis revealed a three-class pattern. The measurement invariance indicated that the instrument can be used among different age groups as well as among males and females. CONCLUSION: The Chinese version of the PIT is a reliable and valid tool to measure prospective imagery, and the positive subscale is meaningful for clinical psychology. Limitations and future research directions are discussed.

Pharmacokinetics, Tissue Distribution, and Druggability Prediction of the Natural Anticancer Active Compound Cytisine N-Isoflavones Combined with Computer Simulation.

Cytisine N-methylene-(5,7-dihydroxy-4'-methoxy)-isoflavone (CNF2) is a new compound isolated from the Chinese herbal medicine Sophora alopecuroides. Preliminary pharmacodynamic studies demonstrated its activity in inhibiting breast cancer cell metastasis. This study examined the pharmacokinetics, absolute bioavailability, and tissue distribution of CNF2 in rats, and combined computer-aided technology to predict the druggability of CNF2. The binding site of CNF2 and the breast cancer target human epidermal growth factor receptor-2 (HER2) were examined with molecular docking technology. Next, ACD/Percepta software was used to predict the druggability of CNF2 based on the quantitative structure-activity relationship (QSAR). Finally, a simple and effective HPLC method was used to determine plasma pharmacokinetics and tissue distribution of CNF2 in rats. Prediction and experimental results show that compared with the positive control HER2 inhibitor SYR127063, CNF2 has a stronger binding affinity with HER2, suggesting that its efficacy is stronger; and the structure of CNF2 complies with the Lipinski's Rule of Five and has good drug-likeness. The residence time of CNF2 in rats is less than 4 h, and the metabolic rate is relatively fast; But the absolute bioavailability of CNF2 in rats was 6.6%, mainly distributed in the stomach, intestine, and lung tissues, where the CNF2 contents were 401.20, 144.01, and 245.82 µg/g, respectively. This study constructed rapid screening and preliminary evaluation of active compounds, which provided important references for the development and further research of such compounds.

Synthesis of α-glucosidase-immobilized nanoparticles and their application in screening for α-glucosidase inhibitors.

In order to rapidly and accurately screen compounds present in Chinese herbal medicines for α-glucosidase inhibition activity, we have developed a screening strategy which couples the immobilized enzymes with HPLC analysis. First, the core-shell PMMA/CS nanoparticle carrier was prepared by graft polymerization method. Subsequently, α-glucosidase was loaded on the nanoparticles to prepare the immobilized enzyme and construct a screening model. Secondly, the morphology and structure of the core-shell PMMA/CS material were characterized by transmission electron microscopy (TEM) and infrared spectroscopy (IR). The results indicate that the size distribution was uniform for the α-glucosidase-immobilized nanoparticles, with an average particle size of about 239nm. Finally, the α-glucosidase-immobilized nanoparticles were used to screen α-glucosidase inhibitors from extracts of traditional Chinese medicine and from a mixture of pharmacologically active compounds. Extracts of traditional Chinese medicines were analyzed by HPLC method before and after incubation with the immobilized enzyme nanoparticles. Using this method, magnoflorine was selectively separated from methanol extracts of Magnoliae Officinalis Cortex and genistein was selectively separated from a mixture of isoflavone compounds. Both the two compounds have previously been reported to have α-glucosidase inhibitory activity. These results demonstrate that this screening strategy represents a rapid and highly efficient method for the identification of α-glucosidase inhibitors in complex extracts of Chinese herbal medicines.

[Neuro-protective effects and mechanism of Terminalia chebula extract HZ4 on cerebral infarction models].

To study the effect and possible molecular mechanisms of Terminalia chebula extract HZ4 on focal cerebral infarction in rats, 90 healthy male SD rats were randomly divided into sham-operation group, model group, T. chebula extract HZ4 high dose, middle dose and low dose groups (80, 40, 20 mg•kg ⁻¹â€¢d ⁻¹, ig) and positive control group (Panax notoginseng saponins, PNS 30 mg•kg ⁻¹â€¢d ⁻¹, ig). The focal cerebral infarction models were established by photochemical method. After the rats were administered for 7 consecutive days, neurogenic behavior rating of these rats was done by balance beam test and foot fault test. The cells morphological changes of penumbra in focal cerebral infarction were investigated by HE staining method; the infarct volume was detected by TTC staining. The expression levels of ß-catenin and cyclin D1, the key node genes in Wnt signaling pathway of the focal penumbra tissues were detected via RT-PCR. The results showed that, as compared with the model group, behavioural indicators were improved significantly in the rats of administration groups, and the infarct volume and pathological changes of penumbra tissues were also improved at the same time. Compared with the model group, the expression levels of ß-catenin and cyclin D1 in Wnt signaling pathway were significantly up-regulated in administration groups(P<0.01). This study first confirmed that T. chebula extract HZ4 can decrease infarct volume, improve the sport ability score, and promote rehabilitation of model animals. In addition, it could significant up-regulated the expression levels of ß-catenin and cyclin D1, and the mechanism may be associated with Wnt signaling pathway. The study is innovative to a certain extent.

Correlation study between molecular structure of sesquiterpene lactones and the selective adsorption performance of molecularly imprinted polymers.

We preliminarily report that the structure of template molecules and target components correlates with the selective adsorption performance of molecularly imprinted polymer (MIPs) in sesquiterpene lactones. Template molecules involved three categories of sesquiterpene lactones with distinct ring systems: 5-mem lactone ring atractylenolide III, 7-mem lactone ring dehydrocostus lactone, and 10-mem lactone ring costunolide lactone, of which the conformations were verified by variable-temperature (1)H NMR spectroscopy. Reciprocal MIPs were prepared by precipitation polymerization and employed as selective sorbents in the columns of solid phase extraction (SPE). These columns were further used for enriching the mixed adsorption solution of sesquiterpene lactone ingredients and reference components. Finally, the extract of Radix Aucklandiae, a Chinese medicine herb, was used to verify the efficiency of this method. Our results demonstrate that the steric conformational stability of molecules is associated with the selective adsorption of their corresponding MIPs. We have further observed that the maximum adsorption capacity occurs when the target molecule conformation is consistent with that of the template molecule. The addition of more hydrophilic groups correlates with weaker adsorption of MIPs. Our findings provide important information to help guide the selection of appropriate template molecules for synthesis of MIPs with specific adsorption.

Porous membrane ultrafiltration-A novel method for enrichment of the active compounds from micro-plasma samples.

To enrich the active compounds from plasma samples, a novel and simple method has been developed using a porous membrane envelope based on the ultrafiltration technique combining with high-performance liquid chromatography. The ultrafiltration device is a sealed porous membrane envelope prepared with a polypropylene sheet to effectively separate the active small molecules and large biomolecules, and a sample carrier is held inside the envelope to load plasma samples. The enrichment of hyperoside and isoquercitrin from rat plasma was used as an example. Significant factors of this method, such as membrane types, the desorption solvent, and the desorption time were optimized for the ultrafiltration method. Under the optimal conditions, correlation coefficients of 0.999 and 0.998 were obtained for hyperoside and isoquercitrin, respectively, with a linear range between 0.5 and 100µg/mL. The absolute extraction recoveries from 83.2% to 86.8% were achieved. The detection limits of the method for hyperoside and isoquercitrin were 0.22 and 0.20µg/mL, respectively. Compared with protein precipitation, solid-phase extraction and commercial ultrafiltration membrane methods, our proposed method demonstrates lower detection limits and lower cost for extraction. Also, it consumes less plasma samples and is found to be applicable to biological samples.

[Comparative study on two polymerization methods for preparing ginsenoside Rg1 molecularly imprinted polymer separating materials].

To obtain ginsenoside Rg1 molecularly imprinted polymer (MIP) separating materials with high selectivity, enrichment and adsorption performance through directional separation of ginsenoside Rg1 and analogues. In this study, MIPs were respectively prepared by precipitation polymerization and surface imprinted polymerization. Their adsorption performances were compared. The results showed that ginsenoside Rg1 MIPs prepared by the above two methods had a high adsorption performance to template molecules, with the maximum apparent adsorbing capacity of up to 27.74, 46. 80 mg x g(-1), respectively. Moreover, MIPs prepared by surface imprinted polymerization showed higher adsorption capacity than that by precipitation polymerization. The experimental results indicated that as for ginsenoside Rg1 with higher polarity, MIPs prepared by surface imprinted polymerization showed higher selectivity and adsorption performance, which provides provide important reference for preparing imprinted polymers with good adsorption performance with active molecules with strong polarity.

[Chemical constituents contained in Macropanax rosthornii].

OBJECTIVE: To study active constituents of Macropanax rosthornii in treating rheumatoid arthritis. METHOD: Silica gel column chromatography, preparative HPLC and modern spectrum techniques were applied for a systematic study on chemical constituents contained in M. rosthornii. RESULT: Twelve compounds were separated from M. rosthornii and identified as serratagenic acid (1), serjanic acid (2), betulinic acid (3), 6beta-hydroxy-3-oxo-olean-12-en-28-oic acid (4), 3-O-alpha-L-arabinopyranosyl serratagenic acid (5), 3-O-alpha-L-arabinopyranosyl serratagenic acid-29-methyl ester (6) , 3-O-alpha-L-arabinopyranosyl serratagenic acid-28-O-beta-D-glucopyranosyl ester (7), scopoletin (8), beta-sitosterol (9), daucosterol (10), 3,4-dihydroxybenzoic acid (11), and stearic acid (12). CONCLUSION: Above compounds are separated from M. rosthornii for the first time.

[Application of network pharmacology and high through-put technology on active compounds screening from traditional Chinese medicine].

OBJECTIVE: To solve the difficult in the active compounds screening from traditional Chinese medicines (TCM). METHOD: According to preliminary lab results and related literature, systematic analysis of the high-throughput technology was made. RESULT: Technologies of rapid preparation, high-throughput screening and biochip, together with the thought of drug target network, will contribute to TCM research on active ingredients screening, toxic components exclusion and molecular mechanisms. They are key technologies and ideas for modernization of TCM and research of TCM network pharmacology. CONCLUSION: High throughput technology and network pharmacology-related technologies will provide new ideas and key methods for active compounds screening from TCM.

Effective compounds group of Mongolian prescriptions BAIMAI-SAN protect against peripheral neuropathy in lower limbs of rats through neuro protective effect.

ETHNOPHARMACOLOGICAL RELEVANCE: BAIMAI-SAN prescription is a famous Chinese minority complex prescription used for curing neuropathy. MATERIALS AND METHODS: The Effective Compounds Groups of BAIMAI-SAN (ECGBM) is determined by high through-put screening, and it includes picroside II, verbascose, taurine and ellagic acid and borneol. To research the potential protective effect of ECGBM on the function of peripheral neuropathy, diabetic rats with peripheral neuropathy were induced by streptozotocin and treated with ECGBM (0.1, 0.3, 0.9 mg/kg/day i.g.) for 75 days. Primary cortical neuronal cultures were subjected to high d-glucitol, and treated with ECGBM prophylactically. RESULTS: The administration resulted in reductions in speed of sciatic motor nerve conduction velocity (MNCV), sensory nerve conduction velocity (SNCV) and response speed to pain in the sciatic nerve fiber. Data from primary cortical neuronal cultures experiments indicated that neuronal survival rates were increased, and LDH release was decreased and the loss of neurite length was alleviated in ECGBM group. CONCLUSIONS: It is first report that ECGBM could protect the peripheral neuron in diabetic rat in vivo and in vitro. This activity may be associated with the neuron protective effect.

Optimization of gradient separation for chromatographic fingerprint of herbal medicine: a predictive model combined with grid search.

A four-step strategy is described which is aimed at optimizing the separation parameters of the chromatographic fingerprint of herbal medicines. In the optimization procedure, a model is built to predict the retention time of components. The proposed model shows good retention prediction potency with an average relative deviation of 1.78%. The grid search method is used in optimizing gradient parameters. In addition, an iterative procedure is adopted to further improve the prediction accuracy of the gradient retention time and the quality of the chromatogram. The whole optimization process has been validated by real samples.

A new algorithm of piecewise automated beam search for peak alignment of chromatographic fingerprints.

The alignment of chromatographic signals is an important preprocessing step before further multivariate analysis. This paper presents a method, automated peak alignment by beam search (Auto-PABS), to solve the problem of peak shift in chemical chromatographic fingerprints by piecewise shifting and linearly interpolating. It is characterized by searching an adaptive range for the values of shifting and linearly interpolating of each segment. This search range is estimated by the calculation of fast Fourier transform cross correlation between the sample segment and its corresponding reference segment. Thus, arbitrary peak alignment is avoided when the real peak shifts are unknown in a large data set. Since the maximum of search range is close to the real shift, more accurate beam search is adopted to accomplish the optimization process. Simulated data and herbal medicine fingerprints of HPLC and GC are selected for evaluation. The output matrix of aligned chromatographic profiles is used directly for principal components analysis, yielding satisfactory results on real samples.