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The characterization of monoclonal antibodies (mAbs) requires laborious and time-consuming sample preparation steps before the liquid chromatography–mass spectrometry (LC-MS) analysis. Middle-up approaches entailing the use of specific proteases (papain, IdeS, etc.) emerged as practical and informative methods for mAb characterization. This work reports the development of immobilized enzyme reactors (IMERs) based on papain able to support mAb analytical characterization. Two monolithic IMERs were prepared by the covalent immobilization of papain on different supports, both functionalized via epoxy groups: a Chromolith® WP 300 Epoxy silica column from Merck KGaA and a polymerized high internal phase emulsion (polyHIPE) material synthesized by our research group. The two bioreactors were included in an in-flow system and characterized in terms of immobilization yield, kinetics, activity, and stability using Nα-benzoyl-L-arginine ethyl ester (BAEE) as a standard substrate. Moreover, the two bioreactors were tested toward a standard mAb, namely, rituximab (RTX). An on-line platform for mAb sample preparation and analysis with minimal operator manipulation was developed with both IMERs, allowing to reduce enzyme consumption and to improve repeatability compared to in-batch reactions. The site-specificity of papain was maintained after its immobilization on silica and polyHIPE monolithic supports, and the two IMERs were successfully applied to RTX digestion for its structural characterization by LC-MS. The main pros and cons of the two supports for the present application were described.

Hovenia dulcis Thunberg is an herbal plant, belonging to the Rhamnaceae family, widespread in west Asia, USA, Australia and New Zealand, but still almost unknown in Western countries. H. dulcis has been described to possess several pharmacological properties, such as antidiabetic, anticancer, antioxidant, anti-inflammatory and hepatoprotective, especially in the hangover treatment, validating its use as an herbal remedy in the Chinese Traditional Medicine. These biological properties are related to a variety of secondary metabolites synthesized by the different plant parts. Root, bark and leaves are rich of dammarane-type triterpene saponins; dihydrokaempferol, quercetin, 3,3′,5′,5,7-pentahydroflavone and dihydromyricetin are flavonoids isolated from the seeds; fruits contain mainly dihydroflavonols, such as dihydromyricetin (or ampelopsin) and hovenodulinol, and flavonols such as myricetin and gallocatechin; alkaloids were found in root, barks (frangulanin) and seeds (perlolyrin), and organic acids (vanillic and ferulic) in hot water extract from seeds. Finally, peduncles have plenty of polysaccharides which justify the use as a food supplement. The aim of this work is to review the whole scientific production, with special focus on the last decade, in order to update phytochemistry, biological activities, nutritional properties, toxicological aspect and regulatory classification of H. dulcis extracts for its use in the European Union.

Drypetes klainei Pierre ex Pax is used in Cameroon by Baka people in the wound healing process and for the treatment of burns. In a previous paper we demonstrated the ability of both water (WE) and defatted methanol (DME) extracts to accelerate scratch wound closure in fibroblast cultures, thus validating the traditional use of D. klainey stem bark in the treatment of skin lesions. In this work we carried out a bioassay-guided fractionation of the most active DME, which exhibited in vitro efficacy in accelerating wound healing process, in order to isolate and identify the compound/s responsible for the assessed biological activity. HPLC was used for the metabolite profiling of DME and fractions (analytical) and for the isolation of the bioactive compound (semi-preparative). MS analyses and NMR spectroscopy were used for identifying the isolated compound. The abilities of treatments in accelerating wound healing were studied on murine fibroblasts in terms of cell viability and cell migration (scratch wound-healing assay). The results obtained allowed to unambiguously identify the isolated bioactive compound as nigracin, a known phenolic glycoside firstly isolated and characterized from bark and leaves of Populus nigra in 1967. However, this is the first time that nigracin is identified in the Drypetes genus and that a wound healing activity is demonstrated for this molecule. Specifically, we demonstrated that nigracin significantly stimulates fibroblast growth and improves cell motility and wound closure of fibroblast monolayer in a dose-dependent manner, without any toxicity at the concentrations tested, and is still active at very low doses. This makes the molecule particularly attractive as a possible candidate for developing new therapeutic options for wound care.

Hibiscus sabdariffa L. is a tropical plant belonging to the Malvaceae family. In Kurdistan, the Autonomous Region of Iraq, water infusion of H. sabdariffa calyces is recommended for the treatment of hypotension and the common cold. Three distillation techniques: hydrodistillation (HD), steam distillation (SD), and solvent-free microwave-assisted extraction (SFME) have been compared to obtain the essential oils from calyces. The composition of the extracts was investigated by GC-FID and GC-MS. A total of 62 compounds have been identified, from which 55 components were found in HD distillates (95.75%), 37 components in SFME (96.06%), and 29 in SD (99.63%). Chemometric tools were applied to optimise and evidence the relation between distillation techniques and composition of the obtained essential oils as an investigation for the essential oils commercialisation approach of Hibiscus sabdariffa L. that have been done from a long time using conventional hydrodistillation in the local Herbal and Tea markets in Kurdistan.

This paper focuses on the synthesis of innovative functional materials prepared by the polymerization of high internal phase emulsions (HIPEs) for the bioconjugation of active molecules. By applying a computational method, this paper demonstrates how to produce materials with tailored features, such as pore dimensions (on the nano–micro scale), swelling in water and organic solvents and bulk densities. To this aim, starting from different emulsion compositions, ten materials are synthesised and fully characterized. Six different outputs (i.e. swelling in water and THF, mass loss in water and THF, throat and void diameters) are modeled using D-optimal mixture experimental designs. A clear correlation between the applied synthetic conditions and the final properties of the materials is demonstrated. The responses allow the selection of a single material for the development of a bioreactor prototype. Thus, the selected material is loaded into a glass column and polymerized in situ. An in-column procedure is also used for the covalent binding of horseradish peroxidase (HRP) as a model enzyme. A standard substrate is selected to test the activity of the immobilized enzyme. To conclude, the experimental design models allow one to obtain different materials with different features that could find applications in the fields such as biocatalysis/biochromatography, drug delivery or diagnostics. To the best of our knowledge, for the first time, an acrylate based polyHIPE material is used for the binding of enzymes in the active form and successfully applied under flow conditions.

The interest towards in-flow biocatalytic processes using immobilised enzyme reactors (IMERs) has grown significantly over the past ten years. In an IMER, the enzyme is immobilized on a suitable chromatographic support, where the catalytic reaction occurs. Interfacing IMERs to a second dimension in integrated analytical systems represents a valuable way to monitor reactions with the advantage of automation, reproducibility, and robustness. This review explores the recent achievements on the development and applications of different analytical set-up for the in-line and on-line monitoring of in-flow bioreactions. The preparation of IMERs is briefly described together with the analytical platforms used to study enzymatic substrate specificity, reaction optimization and kinetic parameters. The review is divided in two parts, one devoted to spectroscopic and spectrometric methods and the other to separative methods. The selected examples illustrate that integration of IMERs in analytical platforms may widespread the industrial applications of biocatalyzed reactions.

Biopharmaceutical drugs are large, complex protein molecules derived from living cells. The characterization of the protein-based products is a complicated and challenging task necessary to guarantee their efficacy and safety. A major concern in manufacturing protein biopharmaceuticals is their propensity to form aggregates. The separation of proteins by size-exclusion chromatography (SEC) is a well-established method for the characterization of macromolecules and their aggregates. The demand for increased resolution and faster analysis in SEC has led to the development of small, more rigid fully porous particles and superficially porous particles with different pore sizes. The coupling of SEC with three or four detectors has enhanced protein characterization by generating accurate molecular weights (absolute determination) and by giving insight into the degradation pathways and products associated with aggregation. The intent of this review is to report the continuous developments of SEC stationary phases and their applications on the characterization of intact proteins, their variants or aggregates and protein complexes. Thus, the most recent applications of SEC with the focus on proteins and protein aggregates are reported and discussed.

The Wnt/β-catenin signaling is a conserved pathway that has a crucial role in embryonic and adult life. Dysregulation of the Wnt/β-catenin pathway has been associated with diseases including cancer, and components of the signaling have been proposed as innovative therapeutic targets, mainly for cancer therapy. The attention of the worldwide researchers paid to this issue is increasing, also in view of the therapeutic potential of these agents in diseases, such as Parkinson's disease (PD), for which no cure is existing today. Much evidence indicates that abnormal Wnt/β-catenin signaling is involved in tumor immunology and the targeting of Wnt/β-catenin pathway has been also proposed as an attractive strategy to potentiate cancer immunotherapy. During the last decade, several products, including naturally occurring dietary agents as well as a wide variety of products from plant sources, including curcumin, quercetin, berberin, and ginsenosides, have been identified as potent modulators of the Wnt/β-catenin signaling and have gained interest as promising candidates for the development of chemopreventive or therapeutic drugs for cancer. In this review we make an overview of the nature-derived compounds reported to have antitumor activity by modulating the Wnt/β-catenin signaling, also focusing on extraction methods, chemical features, and bio-activity assays used for the screening of these compounds., Graphical abstract Dysregulation of the Wnt/β-catenin pathway has been associated with various diseases also including cancer. Several molecular components of the signaling have been proposed as innovative targets for cancer therapy, and many nature-derived compounds have been identified as potent modulators of this pathway. Here we make an overview of these compounds that demonstrated antitumor activity.Image 1

This paper deals with the preparation of new poly(High Internal Phase Emulsion) [polyHIPE] materials, templated by varying different preparation parameters. The obtained systems are based on acrylic monomers, polymerized by free radical polymerization initiated by potassium persulfate and the redox initiator N,N,N′,N′-tetramethylethylenediamine (TEMED) to gain a crosslinked system at room temperature. To get a functional material, glycidyl methacrylate (GMA) has been introduced in the polymeric structure. The HIPE system was effectively stabilized by a co-polymer surfactant, while a polysorbate surfactant did not stabilize the emulsion. Water as the internal phase with its content ranging from 80 to 90% was explored. A deep characterization of the materials, including SEM, DSC, TGA, BET, swelling and weight loss was carried out. The developed systems are promising and can find successful applications for analytical and biomedical purposes.

The paper focuses on the preparation of polyacrylate based biomaterials designed as patches for dermal/transdermal drug delivery using materials obtained by the high internal phase emulsion (HIPE) technique. In particular, butyl acrylate and glycidyl methacrylate were selected, respectively, as backbone and functional monomer while two different crosslinkers, bifunctional or trifunctional, were used to form the covalent network. The influence of PEG on the main properties of the materials was also investigated. The obtained materials show a characteristic and interconnected internal structure as confirmed by SEM studies. By an industrial point of view, an interesting feature of this system is that it can be shaped as needed, in any form and thickness. The physiochemically characterized materials showed a tailorable curcumin (model of hydrophobic drugs) drug release, effective mechanical properties and cell viability and resulted neither pro nor anti-angiogenic as demonstrated in vivo by the chick embryo choriallantoic membrane (CAM) assay. Based on these results, the obtained polyHIPEs could be proposed as devices for dermal/transdermal drug delivery and/or for the direct application on wounded skin.

Affinity-based methods using immobilized proteins are important approaches for understanding the interactions between small molecules and biological targets. This review is intended to provide an overview of different affinity based separation methods that have been applied to the study of peroxisome proliferator activated receptors (PPARs). The screening of compound to increase screening rates for synthetic and natural ligands of PPAR are reported. Pros and cons of the approaches in ligand discovery initiatives are discussed.

Phyllanthus muellerianus (Kuntze) Excell (family Euphorbiaceae) stem bark methanol extract inhibited the growth of Clostridium sporogenes and Streptococcus pyogenes, responsible for gas gangrene and suppurative and non suppurative diseases, respectively. After the HPLC fingerprint acquisition a bioguided fractionation of the defatted methanol extract allowed the isolation of six fractions whose activity was evaluated against the two pathogen bacteria. A further purification of the most active fraction afforded a pure compound responsible for the very interesting inhibitory activity against C. sporogenes and S. pyogenes (MIC 0.91 μM, MIC 3.64 μM). (1)H NMR and MS analytical techniques allowed the identification of the bioactive as Nitidine; this quaternary ammonium alkaloid was observed in the genus Phyllanthus for the first time. A study on Nitidine counter ion, performed using energy dispersive spectroscopy (EDS) coupled with scanning electron microscopy (SEM) was also carried out.

PPAR antagonists are ligands that bind their receptor with high affinity without transactivation activity. Recently, they have been demonstrated to maintain insulin-sensitizing and antidiabetic properties, and they serve as an alternative treatment for metabolic diseases. In this work, an affinity-based bioassay was found to be effective for selecting PPAR ligands from the dried extract of an African plant (Diospyros bipindensis). Among the ligands, we identified betulinic acid (BA), a compound already known for its anti-inflammatory, anti-tumour and antidiabetic properties, as a PPARγ and PPARα antagonist. Cell differentiation assays showed that BA inhibits adipogenesis and promotes osteogenesis; either down-regulates or does not affect the expression of a series of adipogenic markers; and up-regulates the expression of osteogenic markers. Moreover, BA increases basal glucose uptake in 3T3-L1 adipocytes. The crystal structure of the complex of BA with PPARγ sheds light, at the molecular level, on the mechanism by which BA antagonizes PPARγ, and indicates a unique binding mode of this antagonist type. The results of this study show that the natural compound BA could be an interesting and safe candidate for the treatment of type 2 diabetes and bone diseases.

Hydrodistillation of Xylopia hypolampra Mildbr. stem bark afforded 39 mg (dry weight basis) of a pale yellow fragrant essential oil; gas chromatography-flame ionization detector and gas chromatogra...

Aqueous, methanolic and ethyl acetate extracts of Piptadeniastrum africanum (Hook f.) Brenan stem bark were tested against fungi and representatives of Gram-positive and Gram-negative bacteria. Aqueous and methanolic extracts showed a maximum activity against the fungus Pyricularia grisea, a weak activity against the bacteria Staphylococcus aureus and Streptococcus mutans, while no activity was observed against Gram-negative bacteria. Ethyl acetate extract resulted inactive against all the tested microorganisms. Phytochemical investigations of the most active methanolic extract highlighted the presence of tannins and saponins. After purification by means of Sephadex LH-20 and RP-18 the tannin fraction resulted more active against bacteria, while the saponin fraction showed a pronounced activity against Pyricularia grisea. Five different strains of Pyricularia grisea were used and results show that the saponin fraction is promisingly active on the whole population (MIC and MFC: 0.03–0.06 mg mL−1). The saponin fraction fungicidal activity was similar to that of Flutriafol (0.01–0.04 mg mL−1), a commercially available fungicide, indicating that P. africanum stem bark extract can be a good candidate for the control of Pyricularia grisea, the most diffused rice blast disease fungus of rice.

A fast and selective capillary electrophoresis method has been developed for the simultaneous determination of the antihypertensive drugs captopril and hydrochlorothiazide and their related impurities in a combined dosage form. Method development was carried out implementing each step of Quality by Design workflow, the new paradigm of quality outlined in International Conference on Harmonisation Guidelines. Captopril is characterized by the lack of a strong chromophore and contains a proline-similar moiety, which gives rise to the presence of interconverting cis-trans isomers and leads to the possible interference between electrophoretic migration and reaction of isomerization. The scouting phase was dedicated to the investigation of several operative modes in order to overcome detection and isomerization issues. The best performances were obtained with sodium cholate-based micellar electrokinetic chromatography with the addition of n-butanol and γ-cyclodextrin. Critical quality attributes were represented by the critical resolution values and by analysis time. Critical process parameters were defined as temperature, voltage, concentration and pH of borate buffer, concentration of sodium cholate, n-butanol and γ-cyclodextrin. Screening experimental design was applied for investigating knowledge space. Response surface methodology pointed out several significant interaction effects, and with Monte-Carlo simulations led to map out the design space at a selected probability level. Robustness testing was carried out and a control strategy based on system suitability tests was defined. The selected working conditions gave a complete separation of the analytes in less than three minutes. The method was validated and applied to the analysis of a real sample of coformulation tablets.

Ethnopharmacological relevance Allanblackia floribunda Oliv. is one of the most commonly used medicinal plant in Cameroon. The stem bark of the plant is traditionally used for its aphrodisiac and antihypertensive properties. Aim of the study To validate the traditional uses of Allanblackia floribunda stem bark ethanol extract through the evaluation of their aphrodisiac and vasorelaxant properties. Materials and methods The extract's ability to increase sexual desire and the frequencies of erection (mount), intromission and prolonged latency of ejaculation were studied on adult male rats. The vasodilator effect was investigated using isolated rat aorta rings. Tests were conducted using fractions obtained by reverse phase column-chromatography (CC), after the acquisition of the HPLC fingerprint of the ethanol extract, resulted the most active in previous studies. Results The CC allowed the isolation of five fractions whose aphrodisiac and vasodilator activities were tested and compared with those of the whole extract. Four compounds were identified and characterized, three of them, Fukugiside, Morelloflavone and Volkensiflavone, are secondary metabolites known to be in Allanblackia floribunda; the fourth, Spicataside, is a biflavonoid glycoside known to be present in the genus Garcinia but never found neither in Allanblackia floribunda nor in Allanblackia genus. The crude ethanolic extract (CEE) induced a relaxation on aorta rings with EC 50 =11±2 μg/mL and Morelloflavone displayed a similar activity with EC 50 =42±6 μg/mL; for all the other compounds only the vasodilation % at the maximum concentration assessable (90 μg/mL) was determined: 30±8 (Fukugiside), 24±6 (Spicataside), 33±4 (Morelloflavone+Volkensiflavone), 47±1 (Volkensiflavone). Regarding the activity on male sexual behaviour, only CEE and Fukugiside showed activity in the 9 parameters evaluated. Conclusions These results may support the traditional uses of Allanblackia floribunda as aphrodisiac plant with antihypertensive properties suggesting the phytocomplex as responsible for the claimed activity.

Euphorbiaceae barks are known to contain an appreciable amount of polyphenolic compounds responsible for several biological activities. Preliminary extraction from Bridelia grandis stem bark afforded high content of polyphenols, determined by spectrophotometric methods such as Folin-Ciocalteu (for total phenols, TP) and n-butanol-HCl (for condensed tannins, CT). A preliminary Plackett-Burman screening design was used to identify the key factors that influence the TP and CT extraction. Between all the variables known to influence the extraction from vegetable matrixes, six were selected; maceration was chosen as traditional extraction methodology. To investigate the effect of solvents and extraction method, methanol, acetone 70% (v/v in water), centrifugation and ultrasound were chosen. A full factorial design 2(3) was applied to optimize the extraction procedure. The responses were obtained analyzing the extracts for their TP and CT contents determined by the above-mentioned spectrophotometric methods. The results confirm that, within the explored domain, the optimum solvent is methanol and the optimum method is one-cycle centrifugation. Finally, it was also compared with the effect of maceration on the considered responses. It has never given results better than centrifugation, whereas in the case of CT it represents an advantage to employ a three-cycle centrifugation instead of one.

Ethnopharmacological relevance Drypetes klainei Pierre ex Pax is used in Cameroon by Baka Pygmies in the wound healing process and for the treatment of burns. Aim of the study To validate the traditional use of D. klainei Pierre ex Pax stem bark extracts through the evaluation of their antimicrobial properties and their ability to improve wound healing process in fibroblast cell cultures. Materials and methods The antimicrobial properties of D. klainei extracts were evaluated against Staphylococcus aureus ATCC 6538, Streptococcus pyogenes ATCC 19615, Escherichia coli ATCC 10536, Candida albicans ATCC 10231, on the basis of the minimum inhibitory concentration (MIC) and the minimum bactericidal–fungicidal concentration (MBC–MFC) by the macrodilution method. The extracts abilities to accelerate wound healing were studied on murine and human fibroblasts in terms of cell viability and migration (scratch wound-healing assay). Results All the extracts were non-toxic against the selected microorganisms at the tested concentrations, and significantly improve wound healing process in vitro, compared to untreated controls. However, the defatted methanol extract was active at lower concentrations, compared to the water extract. Conclusions The ability of both water and defatted methanol extracts to accelerate scratch wound closure in fibroblast cultures may support the traditional use of D. klainei stem bark in the treatment of skin lesions (such as burns) even if no antimicrobial activity was evidenced.

S-phenylmercapturic acid (PMA) is one specific urinary biomarker of low-level benzene exposure. It is used for biological monitoring of benzene-exposed workers in the petrochemical industry and normally ranges from non-measurable to 10 microg/l levels in non-exposed non-smoking subjects. Benzene-exposure caused by workplace or lifestyle sources is frequently accompanied by toluene exposure, which can cause the occurrence of high levels (from 10 mg/l to more than 2000 mg/l) of hippuric acid (HA) in urine. Both solvents are toxic, and benzene is classified as a human carcinogen. The biological monitoring of benzene and toluene is therefore required for preventive care of exposed workers health. In this study a GC-MS method was adopted for measuring urinary PMA, which involved liquid-liquid extraction (LLE) with ethyl acetate from acidified urine and esterification with 0.5 N hydrochloric acid in methanol. The method evidenced a GC effect in a conventional HP-5 (30 m x 0.25 mm i.d., 0.25 microm film-thickness) methyl-phenylsilicone capillary column produced by HA on PMA. The results demonstrate that HA at concentrations as low as 250 mg/l can delay the elution of PMA and labelled internal standard from the column. The recognition and discussion of this particular GC phase soaking effect may be of help for those who are occupied in the determination of PMA and of urinary acidic metabolites by GC.

The diastereoselective synthesis of chiral nonracemic naphthylaminoalcohols has been accomplished. The diastereoisomers, obtained in high enantiomeric excesses, were investigated by 1H NMR and HPLC analyses. The configurational assignment was performed by NOESY 1H NMR spectroscopy. Pharmacological evaluation of the analgesic activity by means of the hot plate test is described.

In the present paper, we report on the synthesis and antinociceptive activity of a new series of N-methyl-arylpyrrolidinols that we designed for a rational structure-activity relationship (SAR) study. The antinociceptive properties were investigated in vivo by the hot plate and formalin tests in mice and control on the locomotory activity was also monitored by the rota rod test. With this aim, the evaluation of the lipophilicity of all compounds was performed by the Daylight computational method in order to better understand the SAR. Interesting properties were proven for the compounds of the entire series.

The racemic mixtures and the enantiomers of dialkylaminoalkylnaphthalenes are described here as novel analgesic agents with potencies similar, or superior to that of morphine. The synthesis and isolation of the pure enantiomers and a study of the absolute configuration are reported. The resolution of racemates was accomplished by preparative liquid chromatography using a Chiralpak AD column. The configurational assignment was performed on the basis of the X-ray crystallographic analysis of (+)-benzyl-(3-hydroxy-3-naphthalen-2-yl-butyl)dimethylammonium bromide and by comparative study of CD and NOESY 1 H NMR spectra of the resolved enantiomers. Pharmacological evaluation of the analgesic activity by means of the hot plate test is described.

The sequential use of solid-supported reagents and scavengers has led to an efficient synthesis of the natural products nornicotine 1, nicotine 2 and further fuctionalised derivatives. Also reported is a diastereoselective route to both enantiomers of nicotine 2.

The phytochemical research based on ethnopharmacology is considered an effective approach in the discovery of novel chemicals entities with potential as drug leads. Plants/plant extracts/decoctions, used by folklore traditions for treating several diseases, represent a source of chemical entities but no information are available on their nature. Starting from this viewpoint, the aim of this review is to address natural-products chemists to the choice of the best methodologies, which include the combination of extraction/sample preparation tools and analytical techniques, for isolating and characterizing bioactive secondary metabolites from plants, as potential lead compounds in the drug discovery process. The work is distributed according to the different steps involved in the ethnopharmacological approach (extraction, sample preparation, biological screening, etc.), discussing the analytical techniques employed for the isolation and identification of compound/s responsible for the biological activity claimed in the traditional use (separation, spectroscopic, hyphenated techniques, etc.). Particular emphasis will be on herbal medicines applications and developments achieved from 2010 up to date.

The diastereoselective synthesis of naphthyl amino alcohols via nucleophilic addition to racemic 1-dimethylamino-2-methylpentan-3-one was studied. The use of the appropriate experimental conditions allowed the synthesis of both diastereoisomers. The relative configurations were established via NOESY experiments.

Optically pure 2-tributylstannyl-N-Boc-1,3-oxazolidine 6 , prepared from the camphor-derived aminoalcohol 5 , was converted to diastereomerically pure 2-acyl derivatives 8 in three steps. Reaction of these ketones with Grignard reagents at −78°C proceeded with high stereoselectivity affording tertiary carbinols which gave 1,2-diols with >96% ee after hydrolysis and reduction of the intermediate α-hydroxy aldehydes. A new deblocking procedure of the t-Boc group is also described.

Correction for ‘Rational design of functionalized polyacrylate-based high internal phase emulsion materials for analytical and biomedical uses’ by Gloria Brusotti et al., Polym. Chem., 2016, DOI: 10.1039/c6py01992g.

Ethnopharmacological relevance Diospyros bipindensis (Gurke) stem bark is used in Cameroon by Baka Pygmies for the treatment of respiratory disorders. Aim of the study To assess the anti-inflammatory, antibacterial and antioxidant properties of constituents from the bark extracts through bioassay-guided fractionation. Materials and methods The anti-inflammatory activity of extracts, fractions and pure compounds was assessed through the inhibition of the pro-inflammatory mediator nuclear factor-kappa B (NF-κB) transcriptional activity and nitric oxide (NO) production. DPPH, ABTS and ORAC assays were used for determining the antioxidant properties. The activity against Streptococcus pneumoniae, Staphylococcus aureus, Streptococcus pyogenes, Escherichia coli and Klebsiella pneumoniae , was evaluated on the basis of the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) by the macrodilution method. Results The water extract showed antimicrobial activity against S. pneumoniae (MIC: 300 μg/ml) and S. pyogenes (MIC: 300 μg/ml). The dichloromethane extract efficiently inhibited NF-κB transcriptional activity and NO production and exhibited significant antioxidant activity in the ORAC assay. An interesting activity was also found against S. pneumoniae (MIC: 200 μg/ml), S. aureus (MIC: 400 μg/ml) and S. pyogenes (MIC: 200 μg/ml). The phytochemical investigation of the dichloromethane extract afforded plumbagin, canaliculatin, ismailin, betulinic acid and 4-hydroxy-5-methyl-coumarin as the main constituents. Plumbagin and ismailin were found to be responsible for the main biological activities observed. Conclusions These results may provide a rational support for the traditional use of Diospyros bipindensis stem bark in the treatment of respiratory disorders, since the anti-inflammatory, antimicrobial and antioxidant compounds isolated from the dichloromethane extract were also present in the traditional water extract.

Ethnopharmacological relevance Phyllanthus muellerianus (Kuntze) Excel (family Euphorbiaceae) stem bark is used in Cameroon by Baka pygmies as a remedy for wound healing and tetanus. Aim of the study To characterize the chemical composition and to evaluate the antimicrobial properties of the essential oil of the plant. Materials and methods The essential oil was extracted from the stem bark by dynamic head space and by hydrodistillation and characterized by GC and GC-MS analyses. The antimicrobial activity was evaluated on the basis of the minimum inhibitory concentration (MIC) and the minimum bactericidal–fungicidal concentration (MBC–MFC) by the micro and macrodilution methods. The following bacteria and fungi were used: Clostridium sporogenes ATCC 3584, Staphylococcus aureus ATCC 6538, Streptococcus mutans ATCC 25175, Streptococcus pyogenes ATCC 19615, Escherichia coli ATCC 10536, Candida albicans ATCC 10231, Candida albicans LM 450, Trichophyton mentagrophytes LM 230, Trichophyton rubrum LM 237, Microsporum canis LM 324. Results The hydrodistillation afforded 0.06% (dry weight basis) of pale yellow oil. Thirty-eight compounds representing 90.69% were identified. The major component (36.40%) was found to be (E)-isoelemicin, identified by comparison of its 1H-NMR experimental data, with literature data. The oil showed good antibacterial activity against Clostridium Sporogenes, Streptococcus mutans and Streptococcus pyogenes with MIC ranging from 13.5 to 126 μg/ml. A weak antifungal activity (MIC 250 μg/ml) was found against Trichophyton rubrum, only. Conclusions The antimicrobial activity and the chemical composition of Phyllanthus muellerianus stem bark essential oil are reported for the first time.

The preparation of enantiomerically pure 2-lithio-1,3-dioxolanes and 2-lithiooxazolidines and their use as formyl anion equivalents in addition reactions to aldehydes are described.

Ethnopharmacological relevance The plants of the genus Phyllanthus (Euphorbiaceae) are widely distributed in most tropical and subtropical countries, and have long been used in folk medicine to treat several diseases. Particularly, Phyllanthus muellerianus (Kuntze) Excell, commonly called “mbolongo” in Cameroon, is used by pygmies baka as a remedy for tetanus and wound infections. Aim of the study To investigate the antimicrobial properties of Phyllanthus muellerianus (Kuntze) Excell (family Euphorbiaceae) stem bark used in Cameroon by baka pygmies as a remedy for wound healing and tetanus. Materials and methods Aqueous and methanol extracts with and without defatting treatment, were prepared and their activity against Clostridium sporogenes ATCC 3584, Staphylococcus aureus ATCC 6538, Streptococcus mutans ATCC 25175, Streptococcus pyogenes ATCC 19615, Escherichia coli ATCC 10536, Candida albicans ATCC 10231, was evaluated on the basis of the minimum inhibitory concentration (MIC) and the minimum bactericidal-fungicidal concentration (MBC-MFC) by the macrodilution method. Results Water extract showed a weak activity against Clostridium sporogenes (MIC 900 μg/mL) and resulted inactive at the tested concentrations against all the other microorganisms. The defatted methanol extract, inactive against Staphylococcus aureus , Escherichia coli , Candida albicans , exhibited a very interesting activity against Clostridium sporogenes and Streptococcus pyogenes (MIC 100 μg/mL and 300 μg/mL, respectively), which seems to validate the use of this plant in pygmies traditional medicine for the treatment of tetanus and wound infections. The activity found against Streptococcus mutans (300 μg/mL), aetiological agent of caries, may suggest a possible use of this plant as natural remedy to prevent dental diseases. Conclusions The activity against streptococci and Clostridium sporogenes ATCC 3584, showed by stem bark extracts of Phyllanthus muellerianus , traditionally used by baka pygmies to treat wound infections and tetanus, is reported for the first time.

Since the early days of combinatorial chemistry solid-phase organic synthesis has been the method of choice for the production of large libraries. Solution-phase synthesis is again gaining importance especially for the synthesis of parallel arrays of smaller, focussed libraries containing single compounds with high degrees of purity. In the field of solution-phase library generation, the use of solid-supported reagents, catalysts and scavengers is emerging as a leading strategy, combining the advantages of both solid-phase organic synthesis (e.g. allowing the employment of an excess of reagent without the need for additional purification steps) and solution-phase chemistry (e.g. the ease of monitoring the progress of the reactions by applying LC-MS, TLC or standard NMR techniques). An account of some of the most recent advances in this area of research will be presented.

Euphorbiaceae barks are known to contain an appreciable amount of polyphenolic compounds responsible for several biological activities. Preliminary extraction from Bridelia grandis stem bark afforded high content of polyphenols, determined by spectrophotometric methods such as Folin-Ciocalteu (for total phenols, TP) and n-butanol-HCl (for condensed tannins, CT). A preliminary Plackett-Burman screening design was used to identify the key factors that influence the TP and CT extraction. Between all the variables known to influence the extraction from vegetable matrixes, six were selected; maceration was chosen as traditional extraction methodology. To investigate the effect of solvents and extraction method, methanol, acetone 70% (v/v in water), centrifugation and ultrasound were chosen. A full factorial design 2(3) was applied to optimize the extraction procedure. The responses were obtained analyzing the extracts for their TP and CT contents determined by the above-mentioned spectrophotometric methods. The results confirm that, within the explored domain, the optimum solvent is methanol and the optimum method is one-cycle centrifugation. Finally, it was also compared with the effect of maceration on the considered responses. It has never given results better than centrifugation, whereas in the case of CT it represents an advantage to employ a three-cycle centrifugation instead of one.

Aim of the study Bridelia grandis (Pierre ex Hutch) (Euphorbiaceae) is a medicinal plant traditionally used in Cameroon by pygmies Baka as a remedy for oral cavity affection. Bioassay-guided stem bark extracts were investigated for their in vitro antimicrobial properties as well as their phytochemical constituents. Materials and methods The first extraction was carried out according to the traditional use. Further extractions were carried out with solvents of different polarity such as methanol (MeOH), ethyl acetate (EtOAc) and mixtures of MeOH–H 2 O. The antimicrobial activity of the extracts against oral Streptococci was evaluated on the basis of the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) by the macrodilution method; the bacterial surface hydrophobicity was also evaluated. Results Water, methanol and mixtures methanol–water extracts, exhibited antibacterial activity with MIC between 0.5 and 2 mg/ml justifying the traditional use of Bridelia grandis stem bark for oral cavity affection. Preliminary phytochemical analysis was performed on the most active extract (methanol) using appropriate tests and well established analytical screening methods, such as TLC and RP-HPLC/DAD. Conclusions The data obtained indicate that tannins constitute the chemical family responsible for the biological activity.

The chiral recognition mechanism of Penicillin G Acylase (PGA) was investigated with a set of 18 new chiral acidic compounds. A series of 2-aryloxyalkyl- and 2-arylalkyl-2-aryloxyacetic acids in which the absolute configuration has been reported to exert a strong influence on pharmacological activity, were synthesized and analysed on PGA-based chiral stationary phase (CSP) and 11 racemates were completely resolved with a mobile phase composed of 50 mM phosphate buffer (pH 7.0). The influence of structural variations of analytes on retention and enantioselectivity was investigated by application of molecular modelling studies. Docking experiments were also carried out to rationalize the observed enantioselective behaviour. The computation approach revealed to be helpful in elucidating the molecular basis of the enantioselectivity observed on PGA-CSP.