Application of a high-resolution in vitro human MDR1-MDCK assay and in vivo studies in preclinical species to improve prediction of CNS drug penetration.

P-glycoprotein (P-gp, MDR1) is expressed at the blood-brain barrier (BBB) and restricts penetration of its substrates into the central nervous system (CNS). In vitro MDR1 assays are frequently used to predict the in vivo relevance of MDR1-mediated efflux at the BBB. It has been well established that drug candidates with high MDR1 efflux ratios (ERs) display poor CNS penetration. Following a comparison of MDR1 transporter function between the MDR1-MDCKI cell line from National Institutes of Health (NIH) and our internal MDR1-MDCKII cell line, the former was found to provide better predictions of in vivo brain penetration than our in-house MDR1-MDCKII cell line. In particular, the NIH MDR1 assay has an improved sensitivity to differentiate the compounds with ERs of <3 in our internal cell line and is able to reduce the risk of false negatives. A better correlation between NIH MDR1 ERs and brain penetration in rat and non-human primate (NHP) was demonstrated. Additionally, a comparison of brain penetration time course of MDR1 substrates and an MDR1 non-substrate in NHP demonstrated that MDR1 interaction can delay the time to equilibrium of drug concentration in the brain with plasma. It is recommended to select highly permeable compounds without MDR1 interaction for rapid brain penetration to produce the maximal pharmacological effect in the CNS with a quicker onset.

Dietary Astragalus polysaccharides ameliorates the growth performance, antioxidant capacity and immune responses in turbot (Scophthalmus maximus L.).

Supplying immunostimulants to aquatic feed has been an effective way to enhance the health of aquatic animals and substitute for antibiotics. In the present study, the potential effects of Astragalus polysaccharides (APS) were evaluated in turbot, Scophthalmus maximus. Two levels of APS (50 and 150 mg/kg) were added to the basal diet (CON) and a 63-day growth trial (initial weight 10.13 ± 0.04 g) was conducted. As the results showed, significant improvement on growth performance in the APS groups were observed. In addition, dietary 150 mg/kg APS significantly increased the total antioxidant capacity (T-AOC), glutathione peroxidase (GSH-PX) and lysozyme activities in liver. Meanwhile, APS diets induced the mRNA expression of toll-like receptors (TLRs) such as tlr5α, tlr5ß, tlr8 and tlr21, while reduced the expression of tlr3 and tlr22. The expression of inflammatory genes myeloid differentiation factor 88 and nuclear factor kappa b p65 and pro-inflammatory cytokines tumor necrosis factor-α and interleukin-1ß were up-regulated in APS groups while the expression of anti-inflammatory cytokine transforming growth factor beta was inhibited. Taken together, the present study indicated that Astragalus polysaccharides could remarkably enhance the growth performance, antioxidant activity and maintain an active immune response in turbot.

Comparison of the in vitro estrogenic activities of compounds from hops (Humulus lupulus) and red clover (Trifolium pratense).

Because the prevailing form of hormone replacement therapy is associated with the development of cancer in breast and endometrial tissues, alternatives are needed for the management of menopausal symptoms. Formulations of Trifolium pratense L. (red clover) are being used to alleviate menopause-associated hot flashes but have shown mixed results in clinical trials. The strobiles of Humulus lupulusL. (hops) have been reported to contain the prenylflavanone, 8-prenylnaringenin (8-PN), as the most estrogenic constituent, and this was confirmed using an estrogen receptor ligand screening assay utilizing ultrafiltration mass spectrometry. Extracts of hops and red clover and their individual constituents including 8-PN, 6-prenylnaringenin (6-PN), isoxanthohumol (IX), and xanthohumol (XN) from hops and daidzein, formononetin, biochanin A, and genistein from red clover were compared using a variety of in vitro estrogenic assays. The IC50 values for the estrogen receptor alpha and beta binding assays were 15 and 27 microg/mL, respectively, for hops and 18.0 and 2.0 microg/mL, respectively, for the red clover extract. Both of the extracts, genistein, and 8-PN activated the estrogen response element (ERE) in Ishikawa cells while the extracts, biochanin A, genistein, and 8-PN, significantly induced ERE-luciferase expression in MCF-7 cells. Hop and red clover extracts as well as 8-PN up-regulated progesterone receptor (PR) mRNA in the Ishikawa cell line. In the MCF-7 cell line, PR mRNA was significantly up-regulated by the extracts, biochanin A, genistein, 8-PN, and IX. The two extracts had EC50 values of 1.1 and 1.9 microg/mL, respectively, in the alkaline phosphatase induction assay. On the basis of these data, hops and red clover could be attractive for the development as herbal dietary supplements to alleviate menopause-associated symptoms.

Liquid chromatography/electrospray tandem mass spectrometry of terpenoid lactones in Ginkgo biloba.

Ginkgo biloba (ginkgo) is one of most frequently used botanical dietary supplements. The bioactive constituents include the terpenoid lactones consisting of bilobalide and the ginkgolides A, B, C and J. A new assay based on high-performance liquid chromatography/electrospray tandem mass spectrometry (LC/MS/MS) was developed for the measurement of the terpenoid lactones in ginkgo products such as leaf powder and extracts. Initially, the MS/MS fragmentation pathways of ginkgolides were investigated to identify abundant fragment ions that might be useful for the sensitive and selective detection of ginkgolides and bilobalide during LC/MS/MS. Then, sample preparation and clean-up procedures were streamlined to maximize throughput by taking advantage of the selectivity of LC/MS/MS detection. Analyte recoveries exceeded 90%, the intra-assay and inter-assay relative standard deviations were <5%, the relative error was <8% and the limits of detection and quantification were 3.6-120 and 11-350 fmol, depending on the analyte that was injected on to the LC column. Therefore, this LC/MS/MS assay facilitated the rapid quantitative analysis of ginkgolides A, B, C and J and bilobalide in ginkgo dietary supplements with excellent recovery, reproducibity, accuracy and sensitivity.

Identification of caffeic acid derivatives in Actea racemosa (Cimicifuga racemosa, black cohosh) by liquid chromatography/tandem mass spectrometry.

Caffeic acid derivatives occurring in black cohosh [Cimicifuga racemosa (L.) Nutt., Actaea racemosa (Ranunculaceae)], some of which may have pharmacological activity, were analyzed using high-performance liquid chromatography (HPLC) electrospray ionization tandem mass spectrometry (ESI-MS/MS) with the aim of developing a methodology for their rapid identification in a complex plant matrix. Based on these studies, structurally characteristic product ions and neutral molecule losses were identified, which were then used during LC/MS/MS with product ion scanning, precursor scanning and constant neutral loss scanning to detect caffeic acid derivatives in a crude extract of black cohosh. Several caffeic acid derivatives were detected, and the identification of six of them were confirmed by comparison with authentic standards including caffeic acid, ferulic acid, isoferulic acid, fukinolic acid, cimicifugic acid A, and cimicifugic acid B. Four other compounds were detected that appeared to be caffeic acid derivatives based on LC/MS/MS retention times, molecular weights, and fragmentation patterns during MS/MS. Since standards were unavailable for these four compounds, they were tentatively identified using LC/MS/MS as cimicifugic acid E, cimicifugic acid F, dehydrocimicifugic acid A, and dehydrocimicifugic acid B. Dehydrocimicifugic acid A and dehydrocimicifugic acid B have not been reported previously to be constituents of black cohosh.

p-Hydroxybenzoic acid alkyl esters in Andrographis paniculata herbs, commercial extracts, and formulated products.

Analysis of two commercial extracts of Andrographis paniculata using high-performance liquid chromatography (HPLC) with photodiode array absorbance detection showed the presence of several unexpected compounds, which were isolated and identified as methyl, ethyl, and propyl esters of p-hydroxybenzoic acid by using high-resolution mass spectrometry and nuclear magnetic resonance. Quantitative analysis using HPLC revealed the presence of 0.22% p-hydroxybenzoic acid methyl ester (methlyparaben) in one commercial extract, and both 0.11% p-hydroxybenzoic acid ethyl ester (ethylparaben) and 0.20% p-hydroxybenzoic acid propyl ester (propylparaben) in a second commercial extract of A. paniculata. Analyses of additional commercial products of A. paniculata in tablet form purchased from Chicago pharmacies also showed the presence of methyl- and ethylparabens. To determine whether these compounds were natural chemical constituents of the plant, pharmacopoeial reference A. paniculata plant powder as well as samples of authenticated A. paniculata plant materials collected from Indonesia, Hong Kong, and mainland China were obtained and analyzed by HPLC-tandem mass spectrometry (LC-MS-MS). LC-MS-MS analyses confirmed the presence of trace concentrations (<0.0008% w/w) of p-hydroxybenzoic acid methyl ester but no p-hydroxybenzoic acid ethyl or propyl esters in these plant samples. The limits of detection of the LC-MS-MS assay for these compounds were 5 pg on-column and 5 ppb in the plant material. The levels of these p-hydroxybenzoic acid esters measured in the commercial products of A. paniculata suggest that they were introduced inadvertently during processing or as artificial additives.

Evaluation of commercial ginkgo and echinacea dietary supplements for colchicine using liquid chromatography-tandem mass spectrometry.

In response to concerns that commercial dietary supplements containing Ginkgo biloba (ginkgo) and Echinacea purpurea, Echinacea angustifolia, or Echinacea pallida (echinacea) might be contaminated with colchicine, a highly selective and sensitive assay was developed for colchicine that is based on high-performance liquid chromatography-tandem mass spectrometry (LC-MS-MS). The method utilizes reversed-phase HPLC separation of compounds in a methanolic extract of the dietary supplement or botanical sample followed by positive ion electrospray ionization with collision-induced dissociation and multiple reaction monitoring of three characteristic fragmentation pathways of the protonated molecule of colchicine, m/z 400 --> 358, 400 --> 326, and 400 --> 310. The minimal detectable concentration of colchicine using this assay was 10 pg on-column, which is equivalent to 20 ppb colchicine in a 0.5 g ginkgo leaf sample. The method was validated by analyzing 0.5 g samples spiked with colchicine and determining the recovery. A total of 26 commercial ginkgo and echinacea dietary supplements were purchased from pharmacies in Chicago, IL, and analyzed for colchicine. In contrast to a recent report, no colchicine was detected in any of the samples. In addition, authenticated ginkgo leaves were collected, assayed, and found to contain no colchicine, which is consistent with the botanical literature. On the basis of the results obtained using this new LC-MS-MS assay, which is more sensitive and more selective than previously published methods for colchicine, we find no cause for concern regarding colchicine contamination of ginkgo or echinacea dietary supplements.