Nonlinear liquid-liquid chromatography: Beyond a constant distribution coefficient.

Liquid-liquid chromatography (LLC) is a technique in which the separation of mixture components is achieved due to their different distribution between the two phases of a pre-equilibrated biphasic solvent system. In this work, the LLC operation in the nonlinear range of the distribution isotherm was systematically examined for the first time. The influence of the feed concentration on the elution profiles of a model component (cannabidiol, CBD) was studied in three LLC units of different types and sizes ranging from ∼20 mL to ∼2 L. A series of pulse injections with CBD concentrations varying from 1 to 300 mg/mL was performed with n-hexane/methanol/water 5/4/1 (v/v/v) in descending mode (lower phase as the mobile phase). The elution profiles were simulated using the equilibrium-cell model and an anti-Langmuir-like equation for describing the CBD distribution equilibria. The distribution equilibria equation parameters were fitted to the CBD elution profiles using the peak fitting method. The model was validated and provided good predictions of the CBD elution profiles in the entire concentration range for all three LLC units.

AAPH or Peroxynitrite-Induced Biorelevant Oxidation of Methyl Caffeate Yields a Potent Antitumor Metabolite.

Hydroxycinnamic acids represent a versatile group of dietary plant antioxidants. Oxidation of methyl-p-coumarate (pcm) and methyl caffeate (cm) was previously found to yield potent antitumor metabolites. Here, we report the formation of potentially bioactive products of pcm and cm oxidized with peroxynitrite (ONOO¯), a biologically relevant reactive nitrogen species (RNS), or with α,α'-azodiisobutyramidine dihydrochloride (AAPH) as a chemical model for reactive oxygen species (ROS). A continuous flow system was developed to achieve reproducible in situ ONOO¯ formation. Reaction mixtures were tested for their cytotoxic effect on HeLa, SiHa, MCF-7 and MDA-MB-231 cells. The reaction of pcm with ONOO¯ produced two fragments, an o-nitrophenol derivative, and a new chlorinated compound. Bioactivity-guided isolation from the reaction mixture of cm with AAPH produced two dimerization products, including a dihydrobenzofuran lignan that exerted strong antitumor activity in vitro, and has potent in vivo antimetastatic activity which was previously reported. This compound was also detected from the reaction between cm and ONOO¯. Our results demonstrate the ROS/RNS dependent formation of chemically stable metabolites, including a potent antitumor agent (5), from hydroxycinnamic acids. This suggests that diversity-oriented synthesis using ROS/RNS to obtain oxidized antioxidant metabolite mixtures may serve as a valid natural product-based drug discovery strategy.

Brief overview of solubility methods: Recent trends in equilibrium solubility measurement and predictive models.

Solubility is a crucial physicochemical parameter affecting the whole process of drug discovery and development. Thus, understanding of the methods and concepts to measure and predict this propensity are of utmost importance for the pharmaceutical scientist. Despite their inherent limitations, kinetic solubility screening methods became routine assays by mimicking bioassay conditions and guiding the lead optimization process. In contrast, thermodynamic solubility methods show a clear evolution: miniaturized high throughput assays coupled to analytical techniques such as solid-state characterization, ultra performance liquid chromatography, or polychromatic turbidimetry, have been developed, thereby enabling a more complex physicochemical profiling at the early discovery stage. Solubility prediction still poses a significant challenge at the industrial level. Classification and critical evaluation of recent in silico models are provided. Discussion of experimental and computational methods: was based on relevant industrial references.

Three newly identified lipophilic flavonoids in Tanacetum parthenium supercritical fluid extract penetrating the Blood-Brain Barrier.

Feverfew (Tanacetum parthenium L.) as a perennial herb has been known for centuries due to its medicinal properties. The main sesquiterpene lactone, parthenolide is considered to be responsible for the migraine prophylactic effect, however the pharmacological benefits of the lipophilic flavonoid components can not be neglected. Supercritical fluid extraction (7% ethanol, 22MPa, 64°C) was carried out on the leaves of Tanacetum parthenium L. from which the presence of methylated flavonoids beside parthenolide and other sesquiterpene lactones were indicated by preliminary LC-MS analyses. Specific Parallel Artificial Membrane Permeability Assay (PAMPA) was applied to identify the components capable to cross the Blood-Brain Barrier (BBB). Three lipophilic flavonoids were detected on the acceptor side, that were isolated (Prep-HPLC) and identified as sudachitin, aceronin and nevadensin (LC-MS/MS, NMR). These flavonoids were also characterized individually by PAMPA-BBB model. The presence of sudachitin and nevadensin was proven in the Asteraceae family, but neither of the three flavonoids were reported in Tanacetum parthenium L.

BBB penetration-targeting physicochemical lead selection: Ecdysteroids as chemo-sensitizers against CNS tumors.

The anticancer potential of ecdysteroids, especially their chemo-sensitizing activity has recently gained a substantial scientific interest. A comprehensive physicochemical profiling was performed for a set of natural or semi-synthetic ecdysteroids (N=37) to identify a lead compound against central nervous system (CNS) tumors. Calculated properties, such as lipophilicity (clogP), topological polar surface area (TPSA), brain-to-plasma ratio (clogBB) along with the measured blood-brain barrier specific in vitro permeability (logPe) were evaluated in parallel. Compounds with the highest CNS-availability predicted (clogBB>0.0 and logPe>-6.0) showed moderate to high lipophilicity (clogP=3.89-5.25), relatively low TPSA (94.45Å2), and shared a common apolar 2,3- and 20,22-diacetonide motif (25, 30-33). These ecdysteroids were selected for testing their capacity to sensitize SH-SY5Y neuroblastoma cells to vincristine. All of the five tested compounds exerted a remarkably strong, dose dependent chemo-sensitizing activity: at 2.5-10.0µM ecdysteroids increased the cytotoxic activity of vincristine one to three orders of magnitude in (e.g., from IC50=39.5±2.9nM to as low as 0.056±0.03nM). Moreover, analysis of the combination index (CI) revealed outstanding synergism between ecdysteroids and vincristine (CI50=0.072-0.444). Thus, based on drug-likeness, physchem character and in vitro CNS activity, compound 25 was proposed as a lead for further in vivo studies.

Blood-brain barrier specific permeability assay reveals N-methylated tyramine derivatives in standardised leaf extracts and herbal products of Ginkgo biloba.

The linkage between the central nervous system availability and neuropharmacological activity of the constituents of Ginkgo biloba L. extracts (GBE) is still incomplete. In this study, the in vitro blood-brain barrier (BBB) permeability profile of the standardised GBE was investigated by the parallel artificial membrane permeability assay (PAMPA). Biomarkers, such as terpene trilactones, flavonoid aglycones and ginkgotoxin exerted moderate or good BBB-permeability potential (BBB+), while glycosides and biflavones were predicted as unable to pass the BBB. N-methyltyramine (NMT) and N,N-dimethyltyramine or hordenine (Hor) were identified among BBB+ compounds, while subsequent direct HRMS analysis revealed tyramine (Tyr) and N,N,N-trimethyltyramine or candicine (Can) in GBE as trace constituents. Distribution of Tyr, NMT, Hor and Can was determined by a validated ion-exchange mechanism-based liquid chromatography-electrospray ionisation-mass spectrometry (LC-ESI-MS) method in G. biloba samples, such as herbal drugs and dietary supplements. The total content of the four tyramine derivatives in various GBEs ranged from 7.3 up to 6357µg/g dry extract with NMT and Hor as most abundant ones. Considering the pharmacological activities and the revealed fluctuation in the concentration of the analysed adrenergic protoalkaloids, the presented rapid LC-ESI-MS method is proposed for monitoring of the levels of Tyr, NMT, Hor and Can in G. biloba products.

HPLC-DPPH Screening Method for Evaluation of Antioxidant Compounds in Corylus Species.

Corylus avellana L., C. colurna L. and C. maxima Mill. have been used in traditional medicine for a long time for the treatment of various diseases, such as phlebitis, varicose veins, haemorrhoidal symptoms and eczema. Our previous studies indicated the presence of flavonol-3-O-glycosides, diarylheptanoids and caffeic acid derivatives in the bark and leaf extracts of the three species mentioned above. Comparison of in vitro DPPH scavenging activity of the Corylus extracts prepared with ethyl acetate and methanol to those of well-known antioxidant phenolics was performed. The contribution of certain compounds to the total antioxidant activity of the extracts was investigated by developing a HPLC method coupled to the DPPH radical scavenging assay.

An unexpected advantage of insectivorism: insect moulting hormones ingested by song birds affect their ticks.

Ecdysteroids are important hormones that regulate moulting in arthropods. Three-host ixodid ticks normally moult to the next stage after finishing their blood meal, in the off-host environment. Presumably, three-host ticks that feed on the blood of insectivorous vertebrate hosts can be exposed to high levels of exogenous ecdysteroids causing them to initiate apolysis (the first step of moulting) on the vertebrate host. The aim of the present study was to investigate whether ticks undergo apolysis on insectivorous song birds, and if this phenomenon is associated with the seasonal variation in the availability of moths and with the presence of naturally acquired ecdysteroids in avian blood. During a triannual survey, 3330 hard tick larvae and nymphs were collected from 1164 insectivorous song birds of 46 species. A noteworthy proportion of ticks, 20.5%, showed apolysis. The occurrence of apolytic ticks on birds was correlated with the known seasonality of lepidopteran caterpillars. In addition, 18 blood samples of tick-infested birds were analysed with liquid chromatography - tandem mass spectrometry. Eight samples contained ecdysteroids or their derivatives, frequently in high concentrations, and the presence of these was associated with tick apolysis. In conclusion, naturally acquired ecdysteroids may reach high levels in the blood of insectivorous passerine birds, and will affect ticks (feeding on such blood) by shortening their parasitism.

Biomimetic synthesis and HPLC-ECD analysis of the isomers of dracocephins A and B.

Starting from racemic naringenin ((±)-1), a mixture of dracocephin A stereoisomers 6-(2"-pyrrolidinone-5"-yl)naringenin (±)-2a-d and its regioisomer, dracocephin B 8-(2"-pyrrolidinone-5"-yl)naringenin (±)-3a-d originally isolated from Dracocephalum rupestre, have been synthesized in a one-pot reaction. The separation of 2a-d and 3a-d was achieved by preparative HPLC. The four stereoisomers of each natural product were separated by analytical chiral HPLC and their absolute configuration was studied by the combination of HPLC-ECD measurements and TDDFT-ECD calculations. The synthesized flavonoid alkaloids were further characterized by physicochemical and in vitro pharmacological studies.

Tuning the predictive capacity of the PAMPA-BBB model.

Due to its robustness and versatility, several variations of the blood-brain barrier specific parallel artificial membrane permeability assay (PAMPA-BBB) have been reported in the central nervous system (CNS) drug discovery practice. In this study, the impact of the main assay parameters on the predictive power of PAMPA-BBB was thoroughly investigated with 27, passively BBB-transported drug molecules with in vivo logBB data. The single and combined effects of the following variables were systematically studied and optimized: incubation time and temperature (4 vs. 18h, RT vs. 37°C), type of the read-out (UV-reader vs. HPLC), solvent composition (n-dodecane/n-hexane), lipid concentration (0-10w/v % PBLE), cholesterol content (0-1.66w/v %), and thickness of the lipid membrane, and the DMSO cosolvent content (5-20v/v %), respectively. Based on our results, solvent-driven and lipid-driven mechanisms of diffusion were identified in different assay conditions. Moreover, the analysis of membrane retention (MR%; the mole fraction of solute "lost" to the membrane) data obtained at various membrane compositions (volume of solvent and concentration of phospholipids) revealed the compound-specific nature of this parameter. The optimized conditions for the PAMPA-BBB were the following: 4h incubation at 37°C, detection by HPLC-DAD, iso-pH conditions (pH=7.4) with 5v/v % DMSO content in buffer solutions, and PBLE (10w/v %; without cholesterol) as membrane dissolved in the mixture of n-hexane:n-dodecane 3:1.

First characterisation of flavonoid- and diarylheptanoid-type antioxidant phenolics in Corylus maxima by HPLC-DAD-ESI-MS.

Corylus maxima Mill. (Betulaceae) leaves have been used in traditional medicine both internally and externally, nevertheless phytochemical exploration of the plant remains incomplete. In this study, the in vitro antioxidant activity and polyphenolic composition of the ethyl acetate and methanolic extracts of C. maxima leaves and bark are reported for the first time. The radical scavenging activities of the extracts were investigated by the ABTS and DPPH assays. All the extracts of C. maxima possessed notable antioxidant activity. By mean of a HPLC-DAD-ESI-TOF and a HPLC-DAD-ESI-MS/MS method, altogether twenty-two phenolics were tentatively characterised: one flavan derivative (1), seven flavonol derivatives (4, 6, 12, 13, 16, 20 and 21) and fourteen diarylheptanoids (2, 3, 5, 7-11, 14, 15, 17-19 and 22). The amount of the two main flavonoids - myricetin-3-O-rhamnoside (6) and quercetin-3-O-rhamnoside (13) - and two diarylheptanoids - oregonin (3) and hirsutenone (15) - in the extracts were determined by a validated HPLC-ESI-MS/MS method in multiple reaction monitoring (MRM) mode. Our results showed that C. maxima could be considered as a valuable source of pharmacologically important natural products that might contribute to the revaluation of the phytotherapeutical potential of the plant.

Topical analgesic, anti-inflammatory and antioxidant properties of Oxybaphus nyctagineus: phytochemical characterization of active fractions.

ETHNOPHARMACOLOGICAL RELEVANCE: Oxybaphus nyctagineus (Michx.) Sweet has traditionally been used by several Native American tribes predominantly as a topical anti-inflammatory and analgesic agent. AIM OF THE STUDY: To evaluate the antioxidant, analgesic and anti-inflammatory activity of the extracts prepared from the aerial parts of Oxybaphus nyctagineus and to characterize the major chemical constituents of the bioactive extracts. MATERIALS AND METHODS: Crude polar and apolar extracts (PCE and ACE) of the herb of Oxybaphus nyctagineus were prepared and tested in the models of the CFA-induced hyperalgesia in rat knee and carrageenan-induced paw edema in rat. To identify the active compounds, subfractions were prepared by column chromatography and subjected in vitro assays, such as antioxidant assays (DPPH, peroxynitrite (ONOO-) scavenging), and the LPS-induced IL-1ß release test in human monocytes. Preparative HPLC was employed for the isolation of active substances, while phytochemical analysis was performed by mean of LC-MS/MS and NMR. RESULTS: The topically administered PCE and ACE of Oxybaphus nyctagineus demonstrated a significant analgesic and anti-inflammatory effect in the inflammation animal models. The subfraction A4 of ACE and the subfraction P5 of PCE considerably inhibited the LPS-induced IL-1ß release in human monocytes, while the strongest activity was localized in the subfraction P5 in the antioxidant assays. The HPLC-MS/MS and NMR analysis revealed that 6-methoxyflavonol diglycosides, namely patuletin-3-O-robinobioside (1), 6-methoxykaempferol-3-O-robinobioside (2), spinacetin-3-O-robinobioside (3), and hydroxy-polyenoic fatty acids, namely corchorifatty acid B (4), 9-hydroxy-10E,12Z,15Z-octadecatrienoic acid (9-HOT acid) (5), and 9-hydroxy-10E,12Z-octadecadienoic acid (9-HOD acid) (6) were present in PCE, and in ACE as major compounds. CONCLUSION: The results of this study established a pharmacological evidence for the traditional use of Oxybaphus nyctagineus as an anti-inflammatory agent used topically, and provided data on its phytochemical composition for the first time.

Applicability of a blood-brain barrier specific artificial membrane permeability assay at the early stage of natural product-based CNS drug discovery.

While numerous natural products (NPs) possess activity on central nervous system (CNS) targets, there has been no analytical approach to effectively identify compounds with high brain penetration potential in complex mixtures at the early stage of drug discovery. To overcome this issue, the performance of an in vitro parallel artificial membrane permeability assay for the blood-brain barrier (PAMPA-BBB) for natural products and for plant extracts has been validated and characterized. It was found that the PAMPA-BBB assay preserves its predictive power in the case of natural products and provides high phytochemical selectivity, which enables its use as a unique filtering tool in terms of selecting brain-penetrable compounds from plant extracts. Moreover, the present study has demonstrated that simple modifications in the assay design allow the direct use of PAMPA-BBB filtered samples in a dereplication process, as performed by NMR and LC-MS. The applicability of this procedure was demonstrated using extracts prepared from Tanacetum parthenium, Vinca major, Salvia officinalis, and Corydalis cava, representing different types of chemical diversity and complexity. Thus, the proposed protocol represents a potentially valuable strategy in the NP-based CNS drug discovery environment with a high-throughput screening platform.

pH-gradient PAMPA-based in vitro model assay for drug-induced phospholipidosis in early stage of drug discovery.

In the present study we validated a widely used, high-throughput in vitro permeability model (PAMPA) to be used at the early stage of drug discovery for the phospholipidosis (PLD) prediction of drug-like compounds. Regarding the mechanism of action of PLD, our pH-gradient PAMPA system is the first noncell based model to mimic one-way transport of cationic amphiphilic drugs (CADs) from cytosol to the lysosome. Moreover, due to the fact that PLD can mainly occur in lung, liver, brain, kidney and heart, we have used similar commercially available original tissue-derived lipid fractions (heart, liver, brain), and in the case mimicking membrane of kidney and lung tissue we prepared tissue-mimetic artificial lipid mixtures in house. Metabolism of a drug can change the degree of PLD depending on the physicochemical properties of metabolites and the rate of metabolism. Our data from 57 drugs and 4 metabolites of earlier and 2 metabolites of newly recognized outliers (phenacetin and bupropion) using our pH-gradient PAMPA system show a good correlation with in vivo PLD data. Moreover, predictive ability of our best system, the lung specific pH-gradient PAMPA model was significantly better than widely used in silico models and it was also slightly better than that of the known noncell based models on our selection of compounds. Our pH-gradient PAMPA systems therefore offer mechanistically alternative, accurate and cost-effective screening tools for the early prediction of PLD potential of drug-like compounds.

Antioxidant activity-guided phytochemical investigation of Artemisia gmelinii Webb. ex Stechm.: isolation and spectroscopic challenges of 3,5-O-dicaffeoyl (epi?) quinic acid and its ethyl ester.

Although Artemisia gmelinii Webb. ex Stechm. has long been used in south and south-east Asia to treat many kinds of inflammatory diseases, up until now its bioactivity-coupled phytochemical characterization has not been reported. We identified one fraction of the methanolic extract of A. gmelinii as a hit in our antioxidant screening (DPPH) campaign. In order to identify the active radical scavenger components of the extract, a DPPH-HPLC spiking assay was carried out. Out of six detected known compounds caffeic acid and scopoletin had already been identified in the plant, but four of them, namely chlorogenic acid, 4-O-caffeoylquinic acid, luteolin-7-O-glucoside, and apigenin-7-O-glucoside are first described here. Moreover, the two most active compounds of the mixture, 3,5-O-dicaffeoylquinic acid (7) and its ethyl ester derivative (8) were isolated with preparative HPLC. The spectroscopic identification of 7 and 8 presented a surprising challenge due to literature ambiguities. These questions are discussed in detail.

[Plant extracts in drug discovery: traditional considerations, novel possibilities]. / Növényi extraktumok az eredeti gyógyszerkutatásban: tradicionális érvek, új lehetoségek.

Natural product based drug discovery, especially for strategies, based on plant extracts has several and almost traditional arguments. In practice, however, keeping in mind the interest of cost, the chemical diversity and the biological potential provided by secondary plant metabolites could be reached only by rational and harmonized adoption and application of related disciplines and technologies. New and relevant approaches and solutions are presented in this review regarding the accessible way, starting with the collection and extraction of plant material, via the robust high throughput biological screening (HTS) of extracts, reaching isolation, structure elucidation and development phase of the active principle. Special emphasis was made on applications, achieving HTS-compatible extracts and on on-line analytical technics, speeding up the complete isolation process. In addition, beside reviewing international trends and case studies, the results of the examination of the plant extract library of Gedeon Richter Plc. are published as well.

Discovery of novel human histamine H4 receptor ligands by large-scale structure-based virtual screening.

A structure-based virtual screening (SBVS) was conducted on a ligand-supported homology model of the human histamine H4 receptor (hH4R). More than 8.7 million 3D structures derived from different vendor databases were investigated by docking to the hH4R binding site using FlexX. A total of 255 selected compounds were tested by radioligand binding assay and 16 of them possessed significant [(3)H]histamine displacement. Several novel scaffolds were identified that can be used to develop selective H4 ligands in the future. As far as we know, this is the first SBVS reported on H4R, representing one of the largest virtual screens validated by the biological evaluation of the virtual hits.

Nitrone derivatives of trolox as neuroprotective agents.

Synthesis of nitrone derivatives of trolox is described. Their biological evaluation was performed in vitro for scavenging different free radicals, inhibiting Fe(2+)-induced lipid peroxidation, and in vivo in a permanent middle cerebral artery occlusion model in mice. New compounds exert pharmacological activities comparable to or better than those of trolox or nitrone-type reference compounds.