Jose M. Prieto

Traditionally used Thai medicinal plants: In vitro anti-inflammatory, anticancer and antioxidant activities

AIMS OF THE STUDY In order to assess traditional Thai claims about the therapeutic potential of medicinal plants and to select plants for future phytochemical research, nine plant species with anti-inflammatory uses were selected from Thai textbooks and assessed for their in vitro anti-inflammatory, antiproliferative and antioxidant activities. METHODS Nuclear factor-kappaB (NF-kappaB) inhibitory effects in stably transfected HeLa cells were determined by luciferase assay, and effects on LPS-induced pro-inflammatory mediators prostaglandin E2 (PGE2), interleukin (IL)-6, IL-1beta, and tumour necrosis factor (TNF)alpha in primary monocytes were assessed by ELISA. Cytotoxic activities were examined against HeLa cells, human leukaemia CCRF-CEM cells and the multidrug-resistant CEM/ADR5000 subline using the MTT and XTT tests. However, a redox status has been linked with both inflammation and cancer, antioxidant effects were also assessed using the DPPH, lipid-peroxidation, and Folin-Ciocalteau methods. RESULTS Among all the nine species, Gynura pseudochina var. hispida and Oroxylum indicum showed the most promising NF-kappaB inhibitory effects with the lowest IC(50) values (41.96 and 47.45 microg/ml, respectively). Muehlenbeckia platyclada did not inhibit the NF-kappaB activation but effectively inhibited the release of IL-6, IL-1beta and TNF-alpha with IC(50) values ranging between 0.28 and 8.67 microg/ml. Pouzolzia indica was the most cytotoxic against CCRF-CEM cells and the multidrug-resistant CEM/ADR5000 cells (9.75% and 10.48% viability, at 10 microg/ml, respectively). Rhinacanthus nasutus was the most potent cytotoxicity against HeLa cells (IC(50) 3.63 microg/ml) and showed specific cytotoxicity against the multidrug-resistant CEM/ADR5000 cells (18.72% viability at 10 microg/ml, p<0.0001 when compared to its cytotoxicity against CCRF-CEM cells). Moreover, Oroxylum indicum showed a high level of antioxidant activity by inhibiting lipid-peroxidation (IC(50) 0.08 microg/ml). CONCLUSIONS This study provides in vitro evidence for the use of the Thai plants, most importantly Gynura pseudochina var. hispida, Oroxylum indicum and Muehlenbeckia platyclada as Thai anti-inflammatory remedies and these plants are now a priority for further phytochemical research.

Isolation and Characterization of Bioactive Pro-Peptides with in Vitro Renin Inhibitory Activities from the Macroalga Palmaria palmata

Renin is the initial rate limiting step in the renin angiotensinogen system (RAS). To combat hypertension, various stages of the RAS can be positively affected. The aim of this study was to isolate and characterize renin inhibitory peptides from the red seaweed P. palmata for use in functional foods. Palmaria palmata protein was extracted and hydrolyzed with the food grade enzyme Papain to generate renin inhibitory peptides. Following proteolytic hydrolysis of P. palmata protein, reverse phase-high performance liquid chromatography (RP-HPLC) was employed to enrich for peptides with renin inhibitory activities. Fraction 25 (Fr-25) inhibited renin activities by 58.97% (±1.26) at a concentration of 1 mg/mL. This fraction was further characterized using nano-electrospray ionization quadropole/time-of-flight mass spectrometry (ESI-Q/TOF MS). A number of novel peptide sequences were elucidated, and the parent protein from which they were derived was determined using MS in tandem with protein database searches. All sequences were confirmed using de novo sequencing. The renin inhibitory peptide Ile-Arg-Leu-Ile-Ile-Val-Leu-Met-Pro-Ile-Leu-Met-Ala (IRLIIVLMPILMA) was chemically synthesized and its bioactivity confirmed using the renin inhibitory assay. Other stages of the RAS have recently been inhibited by bioactive peptides sourced from macroalgae, but this is the first study to isolate and characterize renin inhibitory peptides from the macroalgae.

Ethnopharmacy of turkish-speaking cypriots in greater London

For centuries, in the Eastern Mediterranean region, medicinal plant use has been widely accepted as a treatment method for both minor and major diseases. Although some knowledge exists on the use of such medicinal plants within the Greek Cypriot culture and considerable information is available on various regions in Turkey, no detailed ethnopharmaceutical or ethnobotanical studies exist on Turkish‐speaking Cypriots (TSC) both in Cyprus and within one of the largest TSC migrant communities in London, UK.

Direct Metabolic Fingerprinting of Commercial Herbal Tinctures by Nuclear Magnetic Resonance Spectroscopy and Mass Spectrometry

INTRODUCTION Tinctures are widely used liquid pharmaceutical preparations traditionally obtained by maceration of one or more medicinal plants in ethanol-water solutions. Such a process results in the extraction of virtually hundreds of structurally diverse compounds with different polarities. Owing to the large chemical diversity of the constituents present in the herbal tinctures, the analytical tools used for the quality control of tinctures are usually optimised only for the detection of single chemical entities or specific class of compounds. OBJECTIVE In order to overcome the major limitations of the current methods used for analysis of tinctures, a new methodological approach based on NMR spectroscopy and MS spectrometry has been tested with different commercial tinctures. METHODOLOGY Diffusion-edited 1H-NMR (1D DOSY) and 1H-NMR with suppression of the ethanol and water signals have been applied here for the first time to the direct analysis of commercial herbal tinctures derived from Echinacea purpurea, Hypericum perforatum, Ginkgo biloba and Valeriana officinalis. The direct injection of the tinctures in the MS detector in order to obtain the corresponding metabolic profiles was also performed. RESULTS Using both NMR and MS methods it was possible, without evaporation or separation steps, to obtain a metabolic fingerprint able to distinguish between tinctures prepared with different plants. Batch-to-batch homogeneity, as well as degradation after the expiry date of a batch, was also investigated. CONCLUSION The techniques proposed here represent fast and convenient direct analyses of medicinal herbal tinctures.

Interaction of N-methyl-2-alkenyl-4-quinolones with ATP-dependent MurE ligase of Mycobacterium tuberculosis: antibacterial activity, molecular docking and inhibition kinetics

Objectives The aim of this study was to comprehensively evaluate the antibacterial activity and MurE inhibition of a set of N-methyl-2-alkenyl-4-quinolones found to inhibit the growth of fast-growing mycobacteria. Methods Using the spot culture growth inhibition assay, MICs were determined for Mycobacterium tuberculosis H37Rv, Mycobacterium bovis BCG and Mycobacterium smegmatis mc2155. MICs were determined for Mycobacterium fortuitum, Mycobacterium phlei, methicillin-resistant Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa using microplate dilution assays. Inhibition of M. tuberculosis MurE ligase activity was determined both by colorimetric and HPLC methods. Computational modelling and binding prediction of the quinolones in the MurE structure was performed using Glide. Kinetic experiments were conducted for understanding possible competitive relations of the quinolones with the endogenous substrates of MurE ligase. Results The novel synthetic N-methyl-2-alkenyl-4-quinolones were found to be growth inhibitors of M. tuberculosis and rapid-growing mycobacteria as well as methicillin-resistant S. aureus, while showing no inhibition for E. coli and P. aeruginosa. The quinolones were found to be inhibitory to MurE ligase of M. tuberculosis in the micromolar range (IC50 ∼40–200 μM) when assayed either spectroscopically or by HPLC. Computational docking of the quinolones on the published M. tuberculosis MurE crystal structure suggested that the uracil recognition site is a probable binding site for the quinolones. Conclusions N-methyl-2-alkenyl-4-quinolones are inhibitors of mycobacterial and staphylococcal growth, and show MurE ligase inhibition. Therefore, they are considered as a starting point for the development of increased affinity MurE activity disruptors.

Herbal medicines in Brazil: pharmacokinetic profile and potential herb-drug interactions

A plethora of active compounds found in herbal medicines can serve as substrate for enzymes involved in the metabolism of xenobiotics. When a medicinal plant is co-administered with a conventional drug and little or no information is known about the pharmacokinetics of the plant metabolites, there is an increased risk of potential herb-drug interactions. Moreover, genetic polymorphisms in a population may act to predispose individuals to adverse reactions. The use of herbal medicines is rapidly increasing in many countries, particularly Brazil where the vast biodiversity is a potential source of new and more affordable treatments for numerous conditions. Accordingly, the Brazilian Unified Public Health System (SUS) produced a list of 71 plant species of interest, which could be made available to the population in the near future. Physicians at SUS prescribe a number of essential drugs and should herbal medicines be added to this system the chance of herb-drug interactions further increases. A review of the effects of these medicinal plants on Phase 1 and Phase 2 metabolic mechanisms and the transporter P-glycoprotein was conducted. The results have shown that approximately half of these medicinal plants lack any pharmacokinetic data. Moreover, most of the studies carried out are in vitro. Only a few reports on herb-drug interactions with essential drugs prescribed by SUS were found, suggesting that very little attention is being given to the safety of herbal medicines. Here we have taken this information to discuss the potential interactions between herbal medicines and essential drugs prescribed to Brazilian patients whilst taking into account the most common polymorphisms present in the Brazilian population. A number of theoretical interactions are pinpointed but more pharmacokinetic studies and pharmacovigilance data are needed to ascertain their clinical significance.

Effect of provenance, plant part and processing on extract profiles from cultivated European Rhodiola rosea L. for medicinal use

The demand for plant material of Rhodiola rosea L. (Crassulaceae) for medicinal use has increased recently, amid concerns about its quality and sustainability. We have analysed the content of phenylpropanoids (total rosavins) and salidroside in liquid extracts from 3-year old cultivated plants of European origin, and mapped the influence of plant part (rhizome versus root), genotype, drying, cutting, and extraction solvent to chemical composition. Rhizomes contained 1.5-4 times more salidroside (0.3-0.4% dry wt) and total rosavins (1.2-3.0%) than roots. The qualitative decisive phenylpropanoid content in the extracts was most influenced by plant part, solvent, and genotype, while drying temperature and cutting conditions were of less importance. We have shown that R. rosea from different boreal European provenances can be grown under temperate conditions and identified factors to obtain consistent high quality extracts provided that authentic germplasm is used and distinguished between rhizome, roots and their mixtures.

Development of a seaweed derived platelet activating factor acetylhydrolase (PAF-AH) inhibitory hydrolysate, synthesis of inhibitory peptides and assessment of their toxicity using the Zebrafish larvae assay

The vascular inflammatory role of platelet activating factor acetylhydrolase (PAF-AH) is thought to be due to the formation of lysophosphatidyl choline and oxidized non-esterified fatty acids. This enzyme is considered a promising therapeutic target for the prevention of atherosclerosis and there is a need to expand the available chemical templates of PAF-AH inhibitors. This study demonstrated how natural PAF-AH inhibitory peptides were isolated and characterized from the red macroalga Palmaria palmata. The dried powdered alga was hydrolyzed using the food grade enzyme papain, and the resultant peptide containing fraction generated using RP-HPLC. Several oligopeptides were identified as potential PAF-AH inhibitors following bio-guided fractionation, and the amino acid sequences of these oligopeptides were confirmed by Q-TOF-MS and microwave-assisted solid phase de novo synthesis. The most promising PAF-AH inhibitory peptide had the amino acid sequence NIGK and a PAF-AH IC50 value of 2.32 mM. This peptide may constitute a valid drug template for PAF-AH inhibitors. Furthermore the P. palmata hydrolysate was nontoxic when assayed using the Zebrafish toxicity model at a concentration of 1mg/ml.

Antimycobacterials from Lovage Root (Ligusticum officinale Koch)

The n‐hexane extract of Lovage root was found to significantly inhibit the growth of both Mycobacterium smegmatis mc2155 and Mycobacterium bovis BCG, and therefore a bioassay‐guided isolation strategy was undertaken. (Z)‐Ligustilide, (Z)‐3‐butylidenephthalide, (E)‐3‐butylidenephthalide, 3‐butylphthalide, α‐prethapsenol, falcarindiol, levistolide A, psoralen and bergapten were isolated by chromatographic techniques, characterized by NMR spectroscopy and MS, and evaluated for their growth inhibition activity against Mycobacterium tuberculosis H37Rv using the whole‐cell phenotypic spot culture growth inhibition assay (SPOTi). Cytotoxicity against RAW 264.7 murine macrophage cells was employed for assessing their degree of selectivity. Falcarindiol was the most potent compound with a minimum inhibitory concentration (MIC) value of 20 mg/L against the virulent H37Rv strain; however, it was found to be cytotoxic with a half‐growth inhibitory concentration (GIC50) in the same order of magnitude (SI < 1). Interestingly the sesquiterpene alcohol α‐prethapsenol was found to inhibit the growth of the pathogenic mycobacteria with an MIC value of 60 mg/L, being more specific towards mycobacteria than mammalian cells (SI ~ 2). Colony forming unit analysis at different concentrations of this phytochemical showed mycobacteriostatic mode of action. Copyright

Effects of 20-hydroxyecdysone, Leuzea carthamoides extracts, dexamethasone and their combinations on the NF-κB activation in HeLa cells.

Objectives  The plant steroid 20‐hydroxyecdysterone (20E) and 20E‐containing extracts from Leuzea carthamoides (Willd.) DC are sold with claims of anabolic and immunomodulatory effects. Yet their effect on the activation of nuclear factor kappa B (NF‐κB), a key player in immune response and cell fate, and their influence on the NF‐κB‐inhibiting activity of steroidal anti‐inflammatory drugs is still unknown.