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Silybum marianum (L.) Gaertner (Milk thistle) extracts have been widely used for the treatment of liver pathologies and as a hepatoprotectant against alcohol-induced liver diseases and other harmful drug metabolites or toxins. Silymarin, the active fraction of Silybum marianum tinctures, accounts for 70–80% of the plant's hydro-alcoholic extract and consists of a mixture of flavonolignans and a flavonoid. Silybum marianum tinctures are commonly prescribed by herbal practitioners for the treatment of liver diseases. However, previous studies have showed inconsistency in the therapeutic efficacy of Silybum marianum tinctures, which is believed to have arisen from the content variability of silymarin, resulting from lack of standardized and regulated manufacturing processes. This work was conducted to quantify the silymarin content in commercial Silybum marianum tinctures in order to evaluate their quality. In this study, we report the determination of the total silymarin content in eleven different commercial tinctures retailed in the U.K. using a convenient and accurate HPLC-UV method. The tinctures analyzed differed in the ratio between herb and liquid as well as percentage of ethanol used during the extraction process. Our results showed a direct correlation between the silymarin content in tinctures and the alcohol strength. Following our protocol, silymarin could not be detected in tinctures extracted with 25% ethanol. Effective therapeutic doses were found only in tinctures with a concentration ratio herb to liquid 1:1 (kg/L) and an alcoholic content of 70%. To guarantee quality and safety of herbal medicinal products, legal requirements to produce plant-based products under regulatory frameworks are needed.

When exposed to ethanol, Drosophila melanogaster display a variety of addiction‐like behaviours similar to those observed in mammals. Sensitivity to ethanol can be quantified by measuring the time at which 50% of the flies are sedated by ethanol exposure (ST50); an increase of ST50 following multiple ethanol exposures is widely interpreted as development of tolerance to ethanol. Sensitivity and tolerance to ethanol were measured after administration of the gamma‐aminobutyric acid receptor B (GABAB) agonist (SKF 97541) and antagonist (CGP 54626), when compared with flies treated with ethanol alone. Dose‐dependent increases and decreases in sensitivity to ethanol were observed for both the agonist and antagonist respectively. Tolerance was recorded in the presence of GABAB drugs, but the rate of tolerance development was increased by SKF 97451 and unaltered in presence of CGP 54626. This indicates that the GABAB receptor contributes to both the sensitivity to ethanol and mechanisms by which tolerance develops. The data also reinforce the usefulness of Drosophila as a model for identifying the molecular components of addictive behaviours and for testing drugs that could potentially be used for the treatment of alcohol use disorder (AUD).

Probiotics secrete bile salt hydrolase (BSH) which catalyses the deconjugation and excretion of bile salts in the GI tract altering cholesterol metabolism in the liver. Many probiotic preparations include prebiotics to promote probiotic growth but little is understood about how prebiotics affect BSH activity. In this study the ability of probiotic Lactobacilli species to deconjugate bile salts in the presence of various prebiotics was determined by measuring cholic acid release. The kinetic properties of BSH was assessed to determine the impact the prebiotics on bile salt deconjugation. When L. acidophilus NCTC 1723 was incubated with inulin (1 %) there was a significant (p These results confirm that prebiotics are capable of altering BSH activity in vitro. Similar changes in vivo could potentially affect the claimed health benefits of synbiotics particularly where the desired outcome is lowering of serum cholesterol.

Chagas disease is a neglected pathology responsible for about 12,000 deaths every year across Latin America. Although six million people are infected by the Trypanosoma cruzi, current therapeutic options are limited, highlighting the need for new drugs. Here we report the preliminary structure activity relationships of a small library of 17 novel pyridyl sulfonamide derivatives. Analogues 4 and 15 displayed significant potency against intracellular amastigotes with EC50 of 5.4 µM and 8.6 µM. In cytotoxicity assays using mice fibroblast L929 cell lines, both compounds indicated low toxicity with decent selectivity indices (SI) >36 and >23 respectively. Hence these compounds represent good starting points for further lead optimization.

Introduction:Challenges facing the treatment of type 2 diabetes necessitate the search for agents which act via alternative pathways to provide better therapeutic outcomes. Recently, an increasing body of evidence implicates the activation of oestrogen receptors (ERα and ERβ) in the development and treatment of underlying conditions in type 2 diabetes. This article summarizes available evidence for the involvement of oestrogen receptors in insulin secretion, insulin resistance as well as glucose uptake and highlights the potential of ERβ as a therapeutic target.Background:Recent studies indicate an association between the activation of each of the isoforms of ER and recent findings indicate that ERβ shows promise as a potential target for antidiabetic drugs. In vitro and in vivo studies in receptor knockout mice indicate beneficial actions of selective agonists of ERβ receptor and underscore its therapeutic potential.Conclusion:Studies are needed to further elucidate the exact mechanism underlying the role of ERβ activation as a therapeutic approach in the management of type 2 diabetes.

Aims: To investigate the scientific basis for the wound-healing properties of Funtumia elastica (Apocynaceae) leaf extracts using relevant in vitro fibroblast growth stimulation, antimicrobial and DPPH-antioxidant assays. Place and Duration of Study: School of Health, Sports and Bioscience (Bioscience Laboratories), University of East London in the United Kingdom, between July 2007 and May 2010. Methodology: Methanolic extract of the leaves, and petroleum ether, ethyl acetate, n- butanol and aqueous fractions partitioned thereof were tested for antimicrobial activities against common wound pathogens (such as Staphylococcus spp, Pseudomonas aeroginosa and Escherichia coli). The Broth dilution method was used to determine the minimum inhibitory concentrations (MIC) of the extracts and fractions. The antioxidant activities were also determined using a 2, 2-diphenyl-1-picrylhydrazyl (DPPH) free radical assay; whilst the ability to stimulate fibroblast growth was investigated using the MTT (3- [4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) assay. Results: The n-butanol fraction exhibited the greatest overall activities. It stimulated the growth of fibroblast cells by 28%, and showed MIC range of 0.13 - 1.0 mg/mL against the Staphylococci species, P. aeruginosa, Bacillus subtilis and E. coli. The non-polar petroleum ether fraction exhibited MICs greater than 2.0 mg/mL against all the organisms. All the fractions exhibited antioxidant activities greater than or comparable to that of ascorbic acid. Conclusion: Collectively, the antioxidant activity, fibroblast growth stimulation and the antimicrobial activities demonstrated by F. elastica leaf extracts provide some evidence to support the use of the plant to manage wounds in African folklore medicine.

Scutellaria barbata D.Don is a popular Chinese medicinal plant documented to treat cancer patients in traditional Chinese medicine (TCM). A botanical new investigational drug for breast cancer BZL101 (FDA IDN# 59521) was previously developed in the United States from the aqueous extract of the aerial parts from S. barbata. The early phase 1A and 1B clinical trials show its favorable toxicity profiles, good clinical tolerance, and promising efficacy for patients with metastatic breast cancer. To further evidence the phytopharmacology research, drug development, and anticancer use of this herb, a systematic literature review was performed herein on the phytochemistry, pharmacology, and specifically anticancer clinical evidence. A systematic review of the literature on phytochemical and pharmacological properties of the plant related to cancer treatment employed several web-based scientific databases including Wanfang (Chinese), Pubmed, Web of Science, and Elsevier. Key words included Scutellaria barbata, Ban Zhi Lian, cancer, and tumor. Based on critical quality criteria, only 8 out of 69 reports related to clinical studies of cancer patients in China. This review covered the available literature up to July 2019. The anticancer effects of S. barbata can be explained by the presence of various flavonoids and diterpenoids alkaloids. The underlying mechanisms are primarily summarized as cyclin/cyclin-dependent kinase (CDK)-modulated cell cycle arrest and mitochondria-mediated apoptotic death. The highly cancer-cell selective cytotoxicity and detoxifying effects of S. barbata contribute to a favorable clinical profile and enhanced quality of life for the cancer patient, thereby demanding further study as an adjuvant or alternative to conventional chemotherapy. The phytochemical and pharmacological studies reviewed strongly underpin a fundamental understanding of the anticancer activity of S. barbata and support ongoing clinical trials. The further safety verification and clinical trials are expected to progress S. barbata-based development to finally transform the traditional TCM herb S. barbata to the valuable anticancer drug.

Ethanol is a psychoactive substance causing both short- and long-term behavioural changes in humans and animal models. We have used the fruit fly Drosophila melanogaster to investigate the effect of ethanol exposure on the expression of the Gαq protein subunit. Repetitive exposure to ethanol causes a reduction in sensitivity (tolerance) to ethanol, which we have measured as the time for 50% of a set of flies to become sedated after exposure to ethanol (ST50). We demonstrate that the same treatment that induces an increase in ST50 over consecutive days (tolerance) also causes a decrease in Gαq protein subunit expression at both the messenger RNA and protein level. To identify whether there may be a causal relationship between these two outcomes, we have developed strains of flies in which Gαq messenger RNA expression is suppressed in a time- and tissue-specific manner. In these flies, the sensitivity to ethanol and the development of tolerance are altered. This work further supports the value of Drosophila as a model to dissect the molecular mechanisms of the behavioural response to alcohol and identifies G proteins as potentially important regulatory targets for alcohol use disorders.

The accepted role of dietary prebiotics is to improve probiotic survival and growth in the lower intestine, yet few studies have investigated the possibility that prebiotics may offer benefits beyond their role with probiotics. This study investigates the protective effects of the prebiotics inulin and lactulose against cytotoxic and genotoxic effects in HT-29 human adenocarcinoma colon cells caused by human faecal water, bile acids and 4-nitroquinoline-1-oxide (4-NQO). Using the MTT assay, deoxycholic acid, lithocholic acid and faecal water 50% v/v reduced cell viability by 75, 74 and 50% respectively. In the presence of inulin and lactulose (0.5–2.0%) cell survival increased by 100% for deoxycholic acid, by 30% for lithocholic acid and by 40% for faecal water. Both faecal water (50% v/v) and 4-NQO (1–2 g/ml) were found to be highly genotoxic using the SOS chromotest, however incubation with inulin and lactulose (2%) significantly reduced the bacterial assay SOS inducing potency (SOSIP) by 70% and 57% respectively for faecal water and by up to 29 and 74% for 4-NQO. Similar protection against genotoxicity was observed with 4-NQO using the Ames fluctuation test. These tests show that prebiotics, in the absence of probiotics, were able to protect mammalian cells (HT-29 cells) against cytotoxicity and reduce the genotoxicity of known mutagens in bacterial mutation assays. Reproduction of these results in vivo would suggest that the inclusion of prebiotics in diet may directly confer health benefits beyond those associated with probiotic survival and growth.

Rationale Self-titration is well documented in the tobacco literature. The extent to which e-cigarette users (vapers) self-titrate is unknown. Objective This study explored the effects of high and low nicotine strength liquid on puffing topography, nicotine delivery and subjective effects in experienced vapers. Methods Eleven experienced male vapers completed 60 minutes of ad libitum vaping under low (6 mg/mL) and high (24 mg/mL) nicotine liquid conditions in two separate sessions. Measurements included: puffing topography (puff number, puff duration, volume of liquid consumed); and changes in: plasma nicotine levels, craving, withdrawal symptoms, self-reported hit, satisfaction and adverse effects. Results Liquid consumption and puff number were higher, and puff duration longer, in the low nicotine strength condition (all ps < 0.01). The mean difference in nicotine boost from baseline in the low condition was 8.59 (7.52) ng/mL, 16.99 (11.72) ng/mL and 22.03 (16.19) ng/mL at 10, 30 and 60 minutes respectively. Corresponding values for the high condition were 33.77 (34.88) ng/mL, 35.48 (28.31) ng/mL and 43.57 (34.78) ng/mL (ps < 0.05). There were no statistically significant differences between conditions in self-reported craving, withdrawal symptoms, satisfaction, hit or adverse effects. Conclusions Vapers engaged in compensatory puffing with lower nicotine strength liquid, doubling their consumption. Whilst compensatory puffing was sufficient to reduce craving and withdrawal discomfort, self-titration was incomplete with significantly higher plasma nicotine levels in the high condition.

Ethnopharmacological relevance Three important Anglo-Saxon medical texts from the 10th century contain herbal formulations for over 250 plant species, many of which have yet to be evaluated for their phytochemical and/or pharmacological properties. In this study, three native British plants were selected to determine antimicrobial activity relevant to treating bacterial infections and wounds. Materials and methods Several preparations of Agrimonia eupatoria L., Arctium minus (Hill) Bernh. and Potentilla reptans L. were screened for antimicrobial activity against selected Gram-positive and Gram-negative bacteria of relevance in wounds using a 96 well plate microdilution method (200, 40 and 8 μg/mL). Minimum inhibitory concentration (MIC) values were determined for the most potent extracts from 2 to 0.004 mg/mL and HPLC chromatograms examined by multivariate analysis. Principle components analysis (PCA) was used to identify chemical differences between antimicrobial activity of the crude extracts. Results The HPLC–PCA score plots attributed HPLC peaks to the antimicrobial activity with all three plants inhibiting growth of Gram-positive Staphylococcus aureus by >50% in four or more extracts. The first two principal components (PC) represented 87% of the dataset variance. The P. reptans 75% ethanol root extract exhibited the greatest range of activity with MIC50 at 31.25 μg/mL to a total MIC that was also the minimum bactericidal concentration (MBC) at 1 mg/mL. Additionally, the root of P. reptans, inhibited growth of Gram-negative bacteria with the 75% ethanol extract having a MIC50 at 1 mg/mL against Pseudomonas aeruginosa and the decoction a MIC50 at 3.9 μg/mL against Escherichia coli. Conclusions The results indicate a moderate antimicrobial activity against common wound pathogens for P. reptans suggesting it may well have been effective for treating wound and bacterial infections. Anglo-Saxon literary heritage may provide a credible basis for researching new antimicrobial formulations. Our approach encompassing advanced analytical technologies and chemometric models paves the way for systematic investigation of Anglo-Saxon medical literature for further therapeutic indications to uncover knowledge of native British plants, some of which are currently lost to modern Western herbal medicine.

Three of the four major Anglo-Saxon collections reporting medicinal formulations in England from the 10th century, the Old English Herbarium, Bald's Leechbook and the Lacnunga, could contain leads and insights into new medicinal uses. Previous pharmacological studies of medicinal plants mentioned in Anglo-Saxon medical texts suggested that some were effective and led to the identification and isolation of natural compounds. For example, matricin from yarrow Achillea millefolium L., is a proprionic acid analogue that yields chamazulene carboxylic acid with cyclooxygenase-2 activity similar to that of ibuprofen. As we discuss here, multidisciplinary projects could further explore historical texts to discover additional plant metabolites with potential pharmacological applications.

Despite a lack of scientific authentication, Scutellaria baicalensis is clinically used in Chinese medicine as a traditional adjuvant to chemotherapy of lung cancer. In this study, cytotoxicity assays demonstrated that crude ethanolic extracts of S. baicalensis were selectively toxic to human lung cancer cell lines A549, SK-LU-1 and SK-MES-1 compared with normal human lung fibroblasts. The active compounds baicalin, baicalein and wogonin did not exhibit such selectivity. Following exposure to the crude extracts, cellular protein expression in the cancer cell lines was assessed using 2D gel electrophoresis coupled with MALDI-TOF-MS/Protein Fingerprinting. The altered protein expression indicated that cell growth arrest and apoptosis were potential mechanisms of cytotoxicity. These observations were supported by PI staining cell cycle analysis using flow cytometry and Annexin-V apoptotic analysis by fluorescence microscopy of cancer cells treated with the crude extract and pure active compounds. Moreover, specific immunoblotting identification showed the decreased expression of cyclin A results in the S phase arrest of A549 whereas the G 0 /G 1 phase arrest in SK-MES-1 cells results from the decreased expression of cyclin D1. Following treatment, increased expression in the cancer cells of key proteins related to the enhancement of apoptosis was observed for p53 and Bax. These results provide further insight into the molecular mechanisms underlying the clinical use of this herb as an adjuvant to lung cancer therapy.

Determination of pharmacological activity relevant to wound healing of Bridelia ferruginea leaf, a traditional medicine used to treat wounds in rural Nigeria.Aqueous and ethanolic leaf extracts were tested against bacterial species of relevance to wound infections: Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa. The ethanolic extracts were assessed for their ability to stimulate the growth of human dermal fibroblasts (FS5) and protect against damage induced by hydrogen peroxide. Antioxidant activity was also assessed using the DPPH assay.Both aqueous and ethanolic extracts had weak antibacterial activity (MIC470 μg/ml). A significant effect (p0.001) on the growth of FS5 fibroblasts was observed only at a concentration of 5 μg/ml (28% increase), above which the extracts appeared toxic to the cells. The ethanolic extract offered the highest protection against H(2)O(2) damage to FS5 cells, comparable with catalase (82% at 250 μg/ml). The DPPH assay revealed antioxidant activity of the ethanolic leaf extract with IC(50) 12.5±0.3 μg/ml comparable to l-ascorbic acid (7.3±0.1 μg/ml).The antibacterial, modest fibroblast stimulation activity and relatively strong antioxidant activity lend some support to the topical use of Bridelia ferruginea leaf for wound-healing in the traditional medicine of South-western Nigeria.

This study examines students’ reactions to the virtual biosciences laboratory developed in Second Life® (SL) at the University of East London. Final year undergraduates and masters students studying biotechnology took part in a trial of a virtual Polymerase Chain Reaction (PCR) experiment in Second Life and evaluated their experience by anonymous questionnaire. Learning gains were measured at various points during the study using pre- and post-tests, and interaction with demonstrators was monitored and compared during the real life (RL) practical. Both groups showed a significant increase in learning gain over the pre- and post-tests, although no difference in gains between the two groups was detected. However, students who conducted the PCR experiment in SL required significantly less demonstrator assistance during the subsequent RL practical. The SL practical was well received by students, with 92% of participants reporting that they would like to use the system again and many requesting other experiments to be made available in this manner in the future.

Evaluation of the cytotoxicity of an ethanolic root extract of Sideroxylonfoetidissimum subsp. gaumeri (Sapotaceae) revealed activity against the murine macrophage-like cell line RAW 264.7. Systematic bioassay-guided fractionation of this extract gave an active saponin-containing fraction from which four saponins were isolated. Use of 1D ((1)H, (13)C, DEPT135) and 2D (COSY, TOCSY, HSQC, and HMBC) NMR, mass spectrometry and sugar analysis gave their structures as 3-O-(beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl)-28-O-(alpha-L-rhamnopyranosyl-(1-->3)[beta-D-xylopyranosyl-(1-->4)]-beta-D-xylopyranosyl-(1-->4)-alpha-L-rhamnopyranosyl-(1-->2)-alpha-L-arabinopyranosyl)-16alpha-hydroxyprotobassic acid, 3-O-beta-D-glucopyranosyl-28-O-(alpha-L-rhamnopyranosyl-(1-->3)[beta-D-xylopyranosyl-(1-->4)]-beta-D-xylopyranosyl-(1-->4)-alpha-L-rhamnopyranosyl-(1-->2)-alpha-L-arabinopyranosyl)-16alpha-hydroxyprotobassic acid, 3-O-(beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl)-28-O-(alpha-L-rhamnopyranosyl-(1-->3)-beta-D-xylopyranosyl-(1-->4)[beta-D-apiofuranosyl-(1-->3)]-alpha-L-rhamnopyranosyl-(1-->2)-alpha-L-arabinopyranosyl)-16alpha-hydroxyprotobassic acid, and the known compound, 3-O-beta-D-glucopyranosyl-28-O-(alpha-L-rhamnopyranosyl-(1-->3)[beta-D-xylopyranosyl-(1-->4)]-beta-D-xylopyranosyl-(1-->4)-alpha-L-rhamnopyranosyl-(1-->2)-alpha-L-arabinopyranosyl)-protobassic acid. Two further saponins were obtained from the same fraction, but as a 5:4 mixture comprising 3-O-(beta-D-glucopyranosyl)-28-O-(alpha-L-rhamnopyranosyl-(1-->3)-beta-D-xylopyranosyl-(1-->4)[beta-D-apiofuranosyl-(1-->3)]-alpha-L-rhamnopyranosyl-(1-->2)-alpha-L-arabinopyranosyl)-16alpha-hydroxyprotobassic acid and 3-O-(beta-D-apiofuranosyl-(1-->3)-beta-D-glucopyranosyl)-28-O-(alpha-L-rhamnopyranosyl-(1-->3)[beta-D-xylopyranosyl-(1-->4)]-beta-D-xylopyranosyl-(1-->4)-alpha-L-rhamnopyranosyl-(1-->2)-alpha-L-arabinopyranosyl)-16alpha-hydroxyprotobassic acid, respectively. This showed greater cytotoxicity (IC(50)=11.9+/-1.5 microg/ml) towards RAW 264.7 cells than the original extract (IC(50)=39.5+/-4.1 microg/ml), and the saponin-containing fraction derived from it (IC(50)=33.7+/-6.2 microg/ml).

The main theoretical and practical aspects of 1H and 13C NMR spectroscopy as relevant to dereplication strategies for natural product hit discovery from plants are summarized, presenting diverse research applications related to pharmaceutical studies. These include advances in pulse sequences for molecular structural characterization of isolated compounds, 1H NMR spectroscopy metabolomics platforms for screening natural product libraries, miniaturization via microNMR spectroscopy and cryoprobe technologies, hyphenated LC-NMR-MS systems with online solid phase extraction and natural product 1H NMR spectral databases. Keywords: 1H; 13C; molecular structure; dereplication; complex mixtures; natural product libraries; chemometrics; secondary metabolite mapping; LC-NMR-MS; databases

Monitoring the metabolism of (13)C-labelled substrates by biological tissues allows both the rate of metabolism and the relative importance of metabolic pathways to be determined. In this study high-resolution magic-angle spinning (HRMAS) (13)C NMR spectroscopy is assessed as a technique for determining the labelling of metabolites in brain slices. Freshly prepared rat brain slices were superfused in isotonic salt solution containing [1-(13)C] glucose. HRMAS (1)H and (13)C NMR spectra were acquired of the slices ( approximately 10 mg) at 3 degrees C. Using (1)H NMR spectroscopy it was demonstrated that the concentration of key metabolites indicative of metabolic degradation, including N-acetyl aspartate and lactate, did not change significantly across the approximately 11 h time period required for (13)C NMR spectra. The approach produced high-resolution spectra of intact tissue with the labelling patterns of tissues being indicative of both labelling via pyruvate dehydrogenase found in both neuronal and glial cells, and pyruvate carboxylase, found only within glial cells. This approach is a versatile tool for monitoring the compartmentation of metabolites directly, and will also allow the investigation of aqueous and lipid metabolites simultaneously.

Cryogenic cooling of the NMR radio frequency coils and electronics to give greatly enhanced sensitivity is arguably the most significant recent advance in NMR spectroscopy. Here we report the first cryogenic probe built in flow configuration and demonstrate the application to LC-NMR-MS studies. This probe provides superior sensitivity over conventional noncryogenic flow NMR probes, allowing the use of 100 microL of untreated urine (40% less material than previous studies that required preconcentration) and yet revealing drug metabolites hitherto undetected by LC-NMR-MS at 500 MHz. Besides the known sulfate and glucuronide metabolites, previously undetected metabolites of acetaminophen were directly observable in a 15-min on-flow experiment. Simultaneous MS data also provided knowledge on the NMR-silent functional moieties. Further, stop-flow LC-NMR-MS experiments were conducted for greater signal-to-noise ratios on minor metabolites. The cryoflow probe enables the NMR analysis of lower concentrations of metabolites than was previously possible for untreated biofluids. This strategy is generally applicable for samples containing mass-limited analytes, such as those from drug metabolism studies, biomarker and toxicity profiling, impurity analysis, and natural product analysis.

A widely held view in drug metabolism and pharmacokinetic studies is that the initial 1-isomer to 2-isomer step in the intramolecular acyl migration of drug ester glucuronides is irreversible, and that alpha-1-O-acyl isomers do not occur under physiological conditions. We investigated this hypothesis using high-performance liquid chromatography directly coupled to proton nuclear magnetic resonance spectroscopy (HPLC/1H NMR) and mass spectrometry (LC/MS) to probe the migration reactions of S-naproxen beta-1-O-acyl glucuronide, in phosphate buffer at pH 7.4, 37 degrees C. We report the first direct observation of the alpha-1-O-acyl isomer of a drug ester glucuronide (S-naproxen) formed in a biosystem via the facile acyl migration of the corresponding pure beta-1-O-acyl glucuronide. The unequivocal identification of the reactive product was achieved using stopped-flow one-dimensional HPLC/1H NMR and two-dimensional 1H-1H total correlation spectroscopy (1H-1H TOCSY). Parallel LC/ion-trap mass spectrometry yielded the confirmatory glucuronide masses. Moreover, "dynamic" stopped-flow HPLC/1H NMR experiments revealed transacylation of the isolated alpha-1-O-acyl isomer to a mixture of alpha/beta-2-O-acyl isomers; the reverse reaction from the isolated alpha/beta-2-O-acyl isomers to the alpha-1-O-acyl isomer was also clearly demonstrated. This application of "dynamic" stopped-flow HPLC/1H NMR allows key kinetic data to be obtained on a reactive metabolite that would otherwise be difficult to follow by conventional HPLC and NMR methods where sample preparation and off-line separations are necessary. These data challenge the widely held view that the alpha-1-O-acyl isomers of drug ester glucuronides do not occur under physiological conditions. Furthermore, the similar formation of alpha-1-O-acyl isomers from zomepirac and diflunisal beta-1-O-acyl glucuronides has recently been confirmed (Corcoran et al., unpublished results). Such reactions are also likely to be widespread for other drugs that form ester glucuronides in biological systems. Ultimately, the presence of significant quantities of the kinetically labile alpha-1-O-acyl glucuronide isomer may also have toxicological implications in terms of reactivity toward cellular proteins.

We report the application of high-performance liquid chromatography (HPLC) linked to inductively coupled plasma mass spectrometry (ICPMS) and orthogonal acceleration time-of-flight mass spectrometry (oa-TOFMS) for the identification of phase I and II urinary metabolites of diclofenac. The metabolites were separated by reversed-phase HPLC monitored with a UV diode array detector (UV-DAD) after which 90% of the eluent was directed to an ICPMS source, with the remainder going to an oa-TOF mass spectrometer. Compounds containing (35)Cl, (37)Cl and (32)S were detected specifically using ICPMS and identified by oa-TOFMS. The metabolites detected and identified in this way included glucuronic acid and sulfate conjugates, mono- and dihydroxylated and free diclofenac. In addition a previously unreported in vivo metabolite, an N-acetylcysteinyl conjugate of diclofenac, was also characterised. This is the first application of the combination of HPLC/UV-DAD/ICPMS/oa-TOFMS for the investigation of the metabolic fate of chlorinated xenobiotics by direct biofluid analysis.

Many species of plants in African countries are widely used in the rural communities where there is little or no access to modern medicine. However, the safety and effectiveness of these medicinal plants are poorly evaluated. The stem bark of Parkia biglobosa Jacq. and leaves of Ageratum conyzoides Linn. were investigated for their antibacterial and cytotoxic activities. The plant materials were extracted with 95% ethanol, and fractionated with petroleum ether, chloroform and ethyl acetate. The antibacterial effects of the extracts and fractions of the plant materials were assayed on the bacterial cultures of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Methicillin Resistant Staphylococcus aureus (MRSA) and Clostridium perfringes. Ethanol extracts of P. biglobosa and A. conyzoides were screened for cytotoxicity using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay. Two cancer cell lines (SK-MES 1 and SK-LU 1) and one normal cell line (human skin fibroblast cell line, FS5) were used for the screening of the extracts and the fractions obtained. The ethanolic extracts and fractions of P. biglobosa and A. conyzoides showed the best activity against E. coli, S. aureus and MRSA. All fractions of A. conyzoides leaves have no activity against P. aeruginosa. Human lung cancer cell lines (SK-LU 1 and SK-MES 1) and human skin fibroblast cell line (FS5 cells) were treated with various concentrations (3.9μg/ml-2mg/ml) of the extracts and fractions for 24h. SK-MES 1 cells are more susceptible to treatment with the plant fractions. All the fractions of A. conyzoides leaves and the petroleum ether fraction of P. biglobosa were cytotoxic to SK-MES 1 cells, which to some extent may support their traditional inclusion in herbal preparations for treatment of cancer. The overall results provided evidence that the studied plant extracts might be potential sources of new antibacterial and anticancer drug.

Scutellaria baicalensis root is widely used in China as an adjuvant to orthodox chemotherapy of lung cancer. However, functional biomarkers of this plant for anti-lung cancer activity have not yet been reported. We therefore determined the growth inhibition activity by MTT assay of eight solvent extracts of S. baicalensis in the human lung cancer cell line SK-MES-1. This activity was then mapped onto the secondary metabolite profile of crude extracts by principal components analysis (PCA) of proton NMR and HPLC-UV data. NMR- and HPLC-PCA maps revealed highest inhibitory activity for the non-aqueous extracts. The first two components of both maps discriminated extract activity mainly based on the differential content of three compounds, which were then tested individually. The IC(50) values for baicalin (IC(50): 64+/-5 microM), baicalein (IC(50): 80+/-6 microM) and wogonin (IC(50): 39+/-10 microM) were comparable to that of the antineoplastic cisplatin (IC(50): 79+/-16 microM). A partial least squares regression (PLS)-NMR model highly correlated with the corresponding PLS-HPLC model for prediction of inhibition. Secondary metabolite mapping of lung cancer growth inhibitors in crude extracts may be an important first step to qualify Chinese herbal prescriptions required for meaningful clinical trials of such integrated therapies.

The flora of the Yucatan peninsula (Mexico) includes approximately 3000 plant species. Sideroxylon foetidissimum Jacq. subsp. gaumeri (Sapotaceae) is an endemic plant to the Yucatan peninsula; its fruit is edible and local people use the plant for medicinal purposes, although no details on its preparation or application are available [1,2]. A preliminary cytotoxic evaluation of the ethanolic root extract of S. foetidissimum revealed a potent activity against murine macrophage like cell line RAW 264.7 (IC50=39.54±4.11µg/mL). The systematic bioassay-guided fractionation of the extract resulted in the identification of the active saponin-containing fraction (IC50=33.69±6.19µg/mL). Four new triterpenoid saponins and a 1:1 mixture of two saponins were isolated from the active saponin- containing fraction. The evaluation of their cytotoxic activity revealed no activity for the tested pure saponins; however, the 1:1 mixture of saponins showed a potent activity (IC50=11.91±1.49µg/mL). The isolation of the saponins was carried out using semi-preparative HPLC. The structural assignments of the pure saponins were based on 1D (1H and 13C and DEPT-135) and 2D (COSY, HMBC, HSQC and TOCSY) NMR and mass spectrometry analyses. In this presentation, the isolation, identification and cytotoxic activity of the isolated compounds is discussed in more detail.

Purpose. To measure the rosmarinic acid content of eight commercial tinctures derived from fresh (n= 5) and dried (n=3) Melissa officinalis herb. Methods. Rosmarinic acid and the internal standard (esculin) were purchased from Aldrich Chemical Co. The column used was a Luna C18, 5 um (150 x 4.6 mm I.D., Phenomenex) maintained at ambient room temperature. The HPLC system consisted of a Shimadzu SCL-6B controller, Shimadzu LC-6A pumps, Shimadzu SPD-6A UV single wavelength spectrophotometric detector set to 320 nm and Shimadzu SIL-6B autosampler. Gradient elution of the samples and standard were performed using ammonium formate (0.02 M; pH 6.25 at 27 oC; eluent A) and methanol (eluent B). The gradient elution initial conditions were 2% of eluent B with linear gradient to 60% at 30 min, followed by linear gradient to 90% of eluent B at 31 min, this proportion being maintained for 4 min. The column was then returned to the initial condition at 36 min and maintained until the end of the run at 43 min. The flow rate was 1 mL/min. The assay was validated for sensitivity, accuracy and reproducibility. Results. The content of rosmarinic acid in commercial tinctures was significantly higher in the tinctures made from dried plant material (2.96 – 22.18 mg/mL) compared to fresh plant tinctures (

Nuclear magnetic resonance spectroscopy (NMR) is arguably the most versatile analytical platform for complex mixture analysis. Specifically, interfacing liquid chromatography with parallel NMR and mass spectrometry (LC-NMR-MS) gives comprehensive structural data on metabolites of novel drugs in development. Applications in natural product, combinatorial chemistry and drug metabolism studies are reviewed. Microcoil probes and capillary separation methods have enormous potential. Recent innovations to improve NMR detection limits include CryoFlowProbes and on-line solid-phase extraction (LC-SPE-NMR). These state-of-the-art analytical platforms are widely applicable to identifying novel candidate drugs from diverse complex mixtures within a drug discovery strategy.

The reactive metabolite S-naproxen-beta-1-O-acyl glucuronide was purified from human urine using solid phase extraction (SPE) and preparative HPLC. The structure was confirmed by 600 MHz 1H NMR. Directly coupled 600 MHz HPLC-1H NMR was used to assign the peaks in chromatograms obtained when analysing a sample containing S-naproxen aglycone and the 1-, 2-, 3-, and 4-isomers of S-naproxen-beta-1-O-acyl glucuronide in two simple isocratic reversed phase HPLC-systems. Using mobile phase 1 (50 mM formate buffer pH 5.75/acetonitrile 75:25 v/v) the elution order was: 4-O-acyl isomers, beta-1-O-acyl glucuronide, 3-O-acyl isomers, 2-O-acyl isomers, and S-naproxen aglycone. Using mobile phase II (25 mM potassium phosphate pH 7.40/acetonitrile 80:20 v/v) the elution order was: alpha/beta-4-O-acyl isomers, S-naproxen aglycone, beta-1-O-acyl glucuronide, 3-O-acyl isomers, and alpha/beta-2-O-acyl isomers. In both systems the elution order for the 2-, 3- and 4-O-acyl isomers corresponded with previously published results for 2-, 3-, and 4-fluorobenzoic acid glucuronide isomers determined by reversed phase HPLC-1H NMR (U.G. Sidelmann, S.H. Hansen, C. Gavaghan, A.W. Nicholls, H.A.J. Carless, J.C. Lindon, I.D. Wilson, J.K. Nicholson, J. Chromatogr. B Biomed. Appl. 685 (1996) 113-122]. The alpha-1-O-acyl isomer was found to be present at approximately 3% of the initial S-naproxen-beta-1-O-acyl glucuronide concentration in the glucuronide isomer mixture after 6 h of incubation at pH 7.40 and 37 degrees C. In both HPLC systems it eluted just before the beta-1-O-acyl glucuronide well separated from other isomers. Investigators should consider the possible formation of a alpha-1-O-acyl isomer when studying glucuronide reactivity and degradation.

Directly coupled 750 MHz HPLC-1H NMR spectroscopy has been applied to the characterisation of low level metabolites of 3-amino-2-(2-fluorophenoxy)pyridine (AP) and 3-nitro-2-(2-fluorophenoxy)pyridine (NP) in rat microsomes. In stop-flow HPLC-NMR mode, the direct injection of microsomal extracts enabled the separation and characterisation of minor metabolites. NP is converted into AP to an extent of 93.4% and this is further metabolised to 4- and 6-hydroxy-AP (6 and 0.6% respectively). Unequivocal identification of these metabolites was achieved without the use of a radiolabel or synthetic standards and thus demonstrates the applicability of directly coupled HPLC-NMR to metabolite identification in in vitro systems. The potential exists for HPLC-NMR and HPLC-NMR-MS to provide rapid metabolic information within the timescale of high throughput lead optimisation exercises in drug discovery.

Acyl-migrated isomers of drug beta-1-O-acyl glucuronides have been implicated in drug toxicity because they can bind to proteins. The acyl migration and hydrolysis of S-naproxen-beta-1-O-acyl glucuronide (S-nap-g) was followed by dynamic stopped-flow HPLC-1H NMR and HPLC methods. Nine first order rate constants in the chemical equilibrium between six species (S-nap-g, its alpha/beta-2-O-acyl, alpha/beta-3-O-acyl, alpha/beta-4-O-acyl, and alpha-1-O-acyl-migration isomers, and S-naproxen aglycone) were determined by HPLC-UV studies in 25 mM potassium phosphate buffer, pH 7.40, 25 mM potassium phosphate buffer in D2O pD 7.40, and 25 mM potassium phosphate buffer in D2O pD 7.40/MeCN 80:20 v/v (HPLC-1H NMR mobile phase). In the 25 mM potassium phosphate buffer (pH 7.40) the acyl-migration rate constants (h(-1)) were 0.18 (S-nap-g-alpha/beta-2-O-acyl isomer), 0.23 (alpha/beta-2-O-acyl-alpha-1-O-acyl), 2.6 (alpha-1-O-acyl-alpha/beta-2-O-acyl), 0.12 (alpha/beta-2-O-acyl-alpha/beta-3-O-acyl), 0.048 (alpha/beta-3-O-acyl-alpha/beta-2-O-acyl), 0.059 (alpha/beta-3-O-acyl-alpha/beta-4-O-acyl), and 0.085 (alpha/beta-4-O-acyl-alpha/beta-3-O-acyl). The hydrolysis rate constants (h(-1)) were 0.025 (hydrolysis of S-nap-g) and 0.0058 (hydrolysis of all acyl-migrated isomers). D2O and MeCN decreased the magnitude of all nine kinetic rate constants by up to 80%. The kinetic rate constants for the degradation of S-nap-g in the mobile phase used for HPLC-1H NMR determined using HPLC-UV could predict the results obtained by the dynamic stopped-flow HPLC-1H NMR experiments of the individual acyl-migrated isomers. It is therefore recommended that beta-1-O-acyl glucuronide degradation kinetics be investigated by HPLC-UV methods once the identification and elution order of the isomers have been established by HPLC-1H NMR.

High resolution 19F NMR spectroscopy has been successfully applied to the analysis of drug metabolites in whole rat urine from a ‘cassette’ dose study of 6 fluorinated test compounds dosed at 10 mg kg–1 per compound. A total of 21 compound-related molecules were rapidly detected in the 0–8 h urine samples. The metabolic fate of the compounds after cassette administration appeared to be the same as when the compounds were dosed individually. 19F NMR may thus enable metabolism to be estimated for several compounds simultaneously, with minimal sample preparation, and without interference from endogenous molecules in the biofluid. This analytical approach may therefore be of value at the drug discovery stage in the development of ‘high throughput’ in vivo metabolic screens.

Purpose: To compare the flavonoid biomarker content (baicalin, baicalein and wogonin) of eleven commercial tinctures derived from Scutellaria lateriflora aerial parts (n=7) and Scutellaria baicalensis root (n=4). S. lateriflora tinctures are used in by western herbal practitioners to treat anxiety whereas S. baicalensis tinctures are used to treat inflammatory disease. Methods: Baicalin and baicalein were purchased from Aldrich Chemical Co. and Wogonin was purchased from ChromaDex. The internal standard (4-hydroxybenzoic acid) was obtained from Acros Organics. The column used was a Luna C18, 5 ?m (150 x 4.6 mm, Phenomenex) maintained at ambient room temperature. A HP1050 HPLC system was used, comprising a gradient pump with degasser, a variable wavelength UV detector set to 270 nm, and an autosampler. Gradient elution was performed using 0.1% formic acid (eluent A) and methanol (eluent B). The gradient elution initial conditions were 45% B with linear gradient to 60% from 2 to 10 min, followed by linear gradient to 70% B at 30 min, and then linear gradient to 99% B at 31 min, this proportion being maintained for 1 min. The mobile phase was then returned to initial conditions at 33 min and maintained until the end of the run at 35 min. The flow rate was 1 mL/min. The assay was validated for sensitivity, accuracy and reproducibility. Results: The concentration range of biomarkers (baicalin, baicalein and wogonin) in commercial tinctures is reported for S. lateriflora (baicalin: 0-12.66 mg/mL; baicalein: 0-0.63 mg/mL; wogonin: 0-0.16 mg/mL) and for S. baicalensis (baicalin: 0.12-10.61 mg/mL; baicalein: 0.52-5.88 mg/mL; wogonin: 0.08-1.61 mg/mL). Conclusion: The wide variability in biomarker concentrations between commercial tinctures has important implications for the manufacturers of commercial tinctures, for herbal practitioners in the choice of tinctures and not least for pharmacology and clinical researchers.