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Pomace, the press residue from different fruits accumulating as waste product in food industry, contains high amounts of secondary metabolites that could be utilized for health-related applications. This study aims at evaluating the potential of pomaces of apricot, bilberry, and elderberry to serve as a source for endothelial nitric oxide synthase (eNOS)-activating compounds. Five extracts obtained from the lyophilized pomace of apricot and elderberry with solvents of different polarity were found to enhance A23187-stimulated eNOS activity when tested at 50 μg/mL in an [14C]-l-arginine to [14C]-l-citrulline conversion assay in the human endothelium-derived cell line EA.hy926 (p < 0.05). The bioassay-guided fractionation of the extracts obtained with methanol/water (70:30) led to several active fractions from apricot pomace (p < 0.05) and elderberry pomace (p < 0.01). Liquid chromatography–mass spectrometry-based chemical analysis of the extracts and active fractions pointed mainly to triterpenoic acids as active compounds. One particular dihydroxytriterpenoic acid, characteristic for elderberry, was enriched as the main compound in the two most active fractions and might serve as a promising lead structure for further studies.

The traditionally used Central American medicinal plant Pluchea odorata, known as an anti-inflammatory and cancer cell growth-inhibiting remedy, was subjected to bioassay-guided isolation. Structure elucidation by 1D- and 2D-NMR and MS techniques supported by ECD and UV spectroscopic data revealed seven structurally previously undescribed and eight known eudesmane-type sesquiterpenes. Furthermore, one previously undescribed and one known phytol-like alcohol were identified. All compounds were tested for their cytotoxicity in cancer cells and for their anti-invasive effects. Among the eudesmanes, 3α-(2',3'-epoxy-2'-methylbutyryloxy)-4α-hydroxy-11-hydroperoxy-eudesm-6-en-8-one exhibited the most potent cytotoxic activity with an IC50 value of 8.8 μM (after 48 h). Also in an in vitro model measuring the tumor-triggered breaching of the adjacent lymph endothelial cell barrier (3S*,4R*,5S*,10S*,2'R*,3'R*)-3-(2',3'-epoxy-2'-methylbutyryloxy)-4,7-dihydroxy-eudesm-11-en-8-one (IC75 = 47 μM) and (3S*,4R*,5R*,10S*,2'R*,3'R*)-3-(2',3'-epoxy-2'-methylbutyryloxy)-4-acetyloxy-6-methoxy-11-hydroxy-eudesm-6-en-8-one (IC75 = 73 μM) showed inhibitory activities. Furthermore, preliminary structure-activity relationships (SARs) of the eudesmanes were developed.

Apple pomace is a waste product of the apple manufacturing industry that has been in the focus of life sciences as it represents a low-cost source of fruit-derived compounds. High fruit consumption is associated with beneficial health effects, and therefore, apple pomace and its constituents raise therapeutic interest. The present work reviews (i) the chemical constituents of apple pomace, (ii) optimized extraction methods of apple pomace compounds, and (iii) biological activities of apple pomace. Current evidence of apple pomace influence on digestion and metabolism, cholesterol and triglyceride homeostasis, diabetes, and sex hormones is summarized. Furthermore, studies regarding its antioxidative, anti-inflammatory, antiproliferative, antibacterial and antiviral effects are presented. The review concludes that apple pomace is an underutilized waste product of the apple industry with the potential of being processed for its nutritional and pharmaceutical value.

Lymph node metastasis of breast cancer is a clinical marker of poor prognosis. Yet, there exist no therapies targeting mechanisms of intravasation into lymphatics. Herein we report on an effect of the antidyslipidemic drug fenofibrate with vasoprotective activity, which attenuates breast cancer intravasation in vitro, and describe the potential mechanisms. To measure intravasation in a 3-dimensional co-culture model MDA-MB231 and MCF-7 breast cancer spheroids were placed on immortalised lymphendothelial cell (LEC) monolayers. This provokes the formation of circular chemorepellent induced defects (CCIDs) in the LEC barrier resembling entry ports for the intravasating tumour. Furthermore, the expression of adhesion molecules ICAM-1, CD31 and FAK was investigated in LECs by western blotting as well as cell-cell adhesion and NF-κB activity by respective assays. In MDA-MB231 cells the activity of CYP1A1 was measured by EROD assay. Fenofibrate inhibited CCID formation in the MDA-MB231/LEC- and MCF-7/LEC models and the activity of NF-κB, which in turn downregulated ICAM-1 in LECs and the adhesion of cancer cells to LECs. Furthermore, CD31 and the activity of FAK were inhibited. In MDA-MB231 cells, fenofibrate attenuated CYP1A1 activity. Combinations with other FDA-approved drugs, which reportedly inhibit different ion channels, attenuated CCID formation additively or synergistically. In summary, fenofibrate inhibited NF-κB and ICAM-1, and inactivated FAK, thereby attenuating tumour intravasation in vitro. A combination with other FDA-approved drugs further improved this effect. Our new concept may lead to a novel therapy for cancer patients.

Objectives Cognitive processing therapy (CPT) is a psychotherapy for post-traumatic stress disorder (PTSD) with a broad evidence base. Change in trauma-related cognitions is considered its primary working mechanism. When trying to integrate a traumatic event into existing cognitive schemas, the adaptive mechanism is changing the schema (accommodation). However, PTSD patients frequently either change their schemas too much (over-accommodation), or cognitively distort the event (assimilation). We aimed to test the hypothesized connections between the three types of cognition and symptom load. Design This study adds to the literature using 'impact statements', essays on their trauma-related thoughts written by patients at the beginning and end of CPT, to investigate cognitive change and its relationship to symptomatic outcome. Methods We analysed statements written by 31 adolescents and young adults who received developmentally adapted CPT (a longer treatment where CPT is the core component) in a randomized controlled trial. Results As expected, post-CPT statements contained more accommodated and fewer over-accommodated and assimilated clauses than pre-CPT statements. Correlations between cognition frequencies and concurrent symptom load were as expected for assimilation, and, in part, over-accommodation and accommodation. Decreased PTSD and depressive symptoms were correlated with increased accommodated thoughts. For over-accommodation and assimilation, however, expected correlations could not be shown. Conclusions Our results support the notion that cognitive change is an important mechanism of change in CPT in a sample of younger, non-English-speaking clients. Practitioner points Knowledge about the working mechanisms of psychotherapies (in addition to knowledge about their efficacy) is important for psychoeducation, motivating clients, and clinical decision-making. In accordance with the theory behind cognitive processing therapy, young clients' cognitions changed over the course of therapy in the predicted ways. For some types of cognition, the change was related to PTSD and/or depressive symptoms, supporting the notion that cognitive change is an important working mechanism of cognitive processing therapy.

Inflammation is part of numerous pathological conditions, which are lacking satisfying treatment and effective concepts of prevention. A national research network project, DNTI, involving scientists from six Austrian universities as well as several external partners aimed to identify and characterize natural products capable of combating inflammatory processes specifically in the cardiovascular system. The combined use of computational techniques with traditional knowledge, high-tech chemical analysis and synthesis, and a broad range of in vitro, cell-based, and in vivo pharmacological models led to the identification of a series of promising anti-inflammatory drug lead candidates. Mechanistic studies contributed to a better understanding of their mechanism of action and delivered new knowledge on the molecular level of inflammatory processes. Herein, the used approaches and selected highlights of the results of this interdisciplinary project are presented. Graphical abstract

Secondary metabolites from lichens are known for exhibiting various biological effects such as anti-inflammatory, antioxidant and antibacterial activities. Despite this wide range of reported biological effects, their impact on the formation of advanced glycation end products (AGEs) remains vastly unexplored. The latter are known contributors to lifestyle and age-related diseases such as Alzheimer and Parkinson. Moreover, the development of atherosclerosis and arterial stiffness is causally linked to the formation of AGEs. With this in mind, the present work evaluated the inhibitory effects of secondary lichen metabolites on the formation of pentosidine-like AGEs' by using an in vitro, Maillard reaction based, fluorescence assay. Overall, thirty-seven natural and five synthetically modified compounds were tested, eighteen of which exhibiting IC50 values in the range of 0.05 to 0.70 mM. This corresponds to 2 to 32 fold of the inhibitory activity of aminoguanidine. Targeting one major inhibiting mechanism of AGEs formation, all compounds were additionally evaluated on their radical scavenging capacities in an DPPH assay. Furthermore, as both AGEs' formation and hypertension are major risk factors for atherosclerosis, compounds that were available in sufficient amounts were also tested for their vasodilative effects. Overall, and though some of the active compounds were previously reported cytotoxic, present results highlight the interesting potential of secondary lichen metabolites as anti-AGEs and vasodilative agents.

Pomace is an easy-accessible raw material for the isolation of fruit-derived compounds. Fruit consumption is associated with health-promoting effects, such as the prevention of cardiovascular disease. Increased vascular nitric oxide (NO) bioavailability, for example, due to an enhanced endothelial nitric oxide synthase (eNOS) activity, could be one molecular mechanism mediating this effect. To identify compounds from apple (Malus domestica Borkh.) pomace that have the potential to amplify NO bioavailability via eNOS activation, a bioassay-guided fractionation of the methanol/water (70:30) extract has been performed using the (14)C-L-arginine to (14)C-L-citrulline conversion assay (ACCA) in the human endothelium-derived cell line EA.hy926. Phytochemical characterization of the active fractions was performed using the spectrophotometric assessment of the total phenolic content, as well as TLC, HPLC-DAD-ELSD, and HPLC-MS analyses. Eleven triterpenoic acids, of which one is a newly discovered compound, were identified as the main constituents in the most active fraction, accompanied by only minor contents of phenolic compounds. When tested individually, none of the tested compounds exhibited significant eNOS activation. Nevertheless, cell stimulation with the reconstituted compound mixture restored eNOS activation, validating the potential of apple pomace as a source of bioactive components.

Background The t(2;5)(p23;q35) chromosomal translocation results in the expression of the fusion protein NPM/ALK that when expressed in T-lymphocytes gives rise to anaplastic large cell lymphomas (ALCL). In search of new therapy options the dichloromethane extract of the ethnomedicinal plant Neurolaena lobata (L.) R.Br. ex Cass was shown to inhibit NPM/ALK expression. Purpose Therefore, we analysed whether the active principles that were recently isolated and found to inhibit inflammatory responses specifically inhibit growth of NPM/ALK+ ALCL, leukaemia and breast cancer cells, but not of normal cells, and the intravasation through the lymphendothelial barrier. Methods ALCL, leukaemia and breast cancer cells, and normal peripheral blood mononuclear cells (PBMCs) were treated with isolated sesquiterpene lactones and analysed for cell cycle progression, proliferation, mitochondrial activity, apoptosis, protein and mRNA expression, NF-κB and cytochrome P450 activity, 12(S)-HETE production and lymphendothelial intravasation. Results In vitro treatment of ALCL by neurolenin B suppressed NPM/ALK, JunB and PDGF-Rβ expression, inhibited the growth of ALCL cells late in M phase, and induced apoptosis via caspase 3 without compromising mitochondrial activity (as a measure of general exogenic toxicity). Moreover, neurolenin B attenuated tumour spheroid intravasation probably through inhibition of NF-κB and CYP1A1. Conclusion Neurolenin B specifically decreased pro-carcinogenic NPM/ALK expression in ALK+ ALCL cells and, via the inhibition of NF-kB signalling, attenuated tumour intra/extravasation into the lymphatics. Hence, neurolenin B may open new options to treat ALCL and to manage early metastatic processes to which no other therapies exist.

P-glycoprotein (ABCB1, MDR1) is capable of extruding chemotherapeutics outside the cell and its overexpression in certain cancer cells may cause failure of chemotherapy. Many attempts were carried out to identify potent inhibitors of this transporter and numerous compounds were shown to exert inhibitory effects in vitro, but so far none were able to make their way to the clinic due to serious complications. Natural compounds represent a great source of therapeutics, which are believed to be safe and effective. Therefore, we have screened a large library of naturally occurring cardiotonic steroids and their derivatives using high throughput flow cytometry. We were able to identify six compounds capable of modulating P-glycoprotein activity. By using P-glycoprotein ATPase assays, molecular docking in silico studies and resazurin reduction assays, the outcome of this high throughput screening platform has been validated. These novel compounds may serve as candidates to reverse doxorubicin resistance in leukemia cells.

Ethnopharmacological relevance The root bark of Lycium chinense Miller, Lycii Radicis Cortex, has been used in traditional Chinese medicine (TCM) to treat different inflammation-related symptoms, such as diabetes mellitus. The pro-inflammatory transcription factor nuclear factor kappa B (NF-κB) is a key regulator of inflammation, while the transcription factor peroxisome proliferator-activated receptor gamma (PPARγ) is a key modulator of genes involved in diabetes development. To identify putative active compound(s) from Lycii Radicis Cortex inhibiting NF-κB or activating PPARγ. Material and methods Using activity-guided fractionation, six extracts with different polarity, isolated fractions, and purified compounds from Lycii Radicis Cortex were tested for NF-κB inhibition and PPARγ activation in vitro . The structure of the purified compounds was elucidated by NMR and MS techniques. Results The ethyl acetate extract and the methanol extract of Lycii Radicis Cortex suppressed tumor necrosis factor alpha (TNF-α)-induced activation of NF-κB, while the dichloromethane extract activated PPARγ. Nine phenolic amide analogues, including trans - N -( p -coumaroyl)tyramine ( 1 ), trans - N -feruloyltyramine ( 2 ), trans - N -caffeoyltyramine ( 3 ), dihydro- N -caffeoyltyramine ( 4 ), three neolignanamides ( 5 – 7 ), and two lignanamide ( 8, 9 ), were isolated and their inhibitory potential on NF- κ B was determined ( 1–4 were also contained in water decoction). Two of the nine isolated phenolic amides inhibited TNF-α - induced NF-κB activation. Trans - N -caffeoyltyramine was verified as the key component responsible for the NF-κB inhibition with an IC 50 of 18.4 μM in our cell-based test system. Activation of PPARγ was attributed to a palmitic-acid enriched fraction which displayed concentration-dependent effect ablated upon co-treatment with the PPARγ antagonist T0070907. Conclusions Phenolic amides were confirmed as main components from Lycii Radicis Cortex responsible for NF-κB inhibition. Fatty acids were identified as the major plant constituent responsible for the PPARγ activation. Structure-activity relationship analysis suggests that the NF-κB inhibitory activity of trans - N -caffeoyltyramine may be attributed to its Michael acceptor-type structure (α,β-unsaturated carbonyl group). The data of this study contribute to a better understanding of the molecular mechanism of action of Lycii Radicis Cortex extracts in the context of inflammation.

Arbuscular mycorrhizal (AM) fungi penetrate the cortical cells of the roots of vascular plants, and are widely distributed in soil. The formation of these symbiotic bodies accelerates the absorption and utilization of mineral elements, enhances plant resistance to stress, boosts the growth of plants, and increases the survival rate of transplanted seedlings. We studied the effects of various arbuscular mycorrhizae fungi on the growth and development of licorice (Glycyrrhiza glabra). Several species of AM, such as Glomus mosseae, Glomus intraradices, and a mixture of fungi (G. mosseae, G. intraradices, G. cladoideum, G. microagregatum, G. caledonium and G. etunicatum) were used in our study. Licorice growth rates were determined by measuring the colonization rate of the plants by the fungi, plant dry biomass, phosphorus concentration and concentration of secondary metabolites. We established two cloned strains of licorice, clone 3 (C3) and clone 6 (C6) to exclude the effect of genotypic variations. Our results showed that the AM fungi could in fact increase the leaf and root biomass, as well as the phosphorus concentration in each clone. Furthermore, AM fungi significantly increased the yield of certain secondary metabolites in clone 3. Our study clearly demonstrated that AM fungi play an important role in the enhancement of growth and development of licorice plants. There was also a significant improvement in the secondary metabolite content and yield of medicinal compounds from the roots.

Our inability to effectively “drug” targets such as MYC for therapeutic purposes requires the development of new approaches. We report on the implementation of a phenotype-based assay for monitoring MYC expression in multiple myeloma cells. The open reading frame (ORF) encoding an unstable variant of green fluorescent protein was engineered immediately downstream of the MYC ORF using CRISPR/Cas9, resulting in co-expression of both proteins from the endogenous MYC locus. Using fluorescence readout as a surrogate for MYC expression, we implemented a pilot screen in which ~10,000 compounds were prosecuted. Among known MYC expression inhibitors, we identified cardiac glycosides and cytoskeletal disruptors to be quite potent. We demonstrate the power of CRISPR/Cas9 engineering in establishing phenotype-based assays to identify gene expression modulators.

Ethnopharmacological relevance The vast genus Rhododendron includes species that have been used in traditional medicine for the treatment of inflammatory conditions, pain, gastro-intestinal disorders, common cold, asthma, skin disease, etc. Rhododendrons are also well known for their toxicity and some species have been traditionally used as poison. Aim of the review The work reviews and analyses the traditional use, biological activities with the corresponding chemical constituents, and toxicological data on Rhododendron species. The review aims at characterizing the ethnopharmacology of the genus in relation to its toxicity in order to identify the therapeutic potential of Rhododendron species and future directions for research. Methods Data regarding Rhododendron spp. was collected using electronic databases (SciFinder, PubMed, Google Scholar) and library search for selected peer-reviewed articles. Plant taxonomy was validated by the databases The Plant List, Tropicos, eFloras, Flora Iberica and Flora Europaea (RBGE). Additional information on traditional use and botany was obtained from published books. The review encompasses literature, mainly regarding biological activity and toxicological data, from 1898 to the end of December 2012. Results Rhododendrons have been used in Asian, North American and European traditional medicine mainly against inflammation, pain, skin ailments, common cold and gastro-intestinal disorders. In vivo and in vitro testing of plant extracts and isolated compounds determined diverse biological activities including anti-inflammatory, analgesic, anti-microbial, anti-diabetic, insecticidal and cytotoxic activity. Rhododendron spp. can cause intoxications in humans following intake of rhododendron honey or medicinal preparations. The toxicity is due to grayanotoxins, diterpenes which activate voltage-gated sodium channels and lead to gastro-intestinal, cardiac and central nervous system symptoms. Conclusion Rhododendron species are useful traditional remedies for the treatment of inflammation, pain, skin ailments, common cold and gastro-intestinal disorders. Pharmacological data has validated most indications of rhododendrons in ethnomedicine and toxicology studies have confirmed the toxicity observed by traditional use. Ethnopharmacological data point to the therapeutic potential of the genus Rhododendron for the treatment of inflammatory conditions and pain and, thus, research should focus on identification of active compounds and related mechanistic studies. Prolonged and high dose intake of traditional formulations containing rhododendrons should be avoided until more in depth toxicity studies become available.

Sphingolipid metabolites regulate cellular processes such as cell proliferation, differentiation, and apoptosis. In this study, glucocerebrosides (GluCer) from rhizomes of Arisaema amurense and Pinellia ternata were fully characterized using 1- and 2-dimensional nuclear magnetic spin resonance (NMR) and circular dichroism (CD) spectroscopy and tandem collision-induced dissociation mass spectrometry (ESI-MS/CID-MS). Three new acylated and seven known GluCer were elucidated with 4,8-sphingadienine (4,8-SD, d18:2) as backbone. 4,8-SD is a metabolite after enzymatical hydrolysis of GluCer in the gut lumen. In this study, 4,8-SD was hydrolyzed from GluCer and chromatographically purified on silica gel. In contrast to the GluCer, 4,8-SD showed cytotoxic effects in the WST-1 assay. GluCer with 4,8-SD as sphingoid backbone are present in plants consumed as food, such as spinach, soy, and eggplant.

The gamma-amino butyric acid (GABA) type A (GABA(A)) receptor represents a crucial target for clinical agents in the treatment of anxiety and insomnia. Using the two-microelectrode voltage clamp technique on recombinant α₁β₂γ(2S) GABA (A) receptors, effusol (1) and dehydroeffusol (2) were isolated in a bioactivity-guided approach from the pith of Juncus effusus L. Both compounds concentration-dependently enhanced GABA induced chloride currents (I(GABA)) by a maximum 188 ± 20 (1) and 239 ± 18 % (2), independent of the benzodiazepine (BZ) binding site. This activity on the GABA (A) receptor may explain the traditional use of J. effusus as a sedative and anxiolytic agent in Chinese medicine.

Abnormal Savda Munziq(ASMq) is a herbal preparation used in Traditional Uighur Medicine for the treatment and prevention of diabetes, cardiovascular diseases, chronic asthma and cancer. The recommended dose of this decoction for cancer patients is 500 mL administered orally three times a day. Our approach aimed at reducing the high amount of fluid intake required by fractionation of ASMq guided by the antiproliferative activity on HL-60 cells. The fractionation of ASMq resulted in the preparation of an active extract, Extr-4. Using solid phase extraction, Extr-4 was further fractionated into five fractions (SPE-0, SPE-20, SPE-40, SPE-60 and SPE-80), with SPE-40 showing the strongest antiproliferative activity. Caffeic acid, rutin, isoquercitrin, isorhamnetin 3-O-rutinoside, apigenin 7-O-glucoside, rosmarinic acid, luteolin and formononetin were identified in Extr-4 and fractions thereof by means of TLC, HPLC-DAD and LC-MS. SPE-40 contained the main compounds responsible for the antiproliferative activity on HL-60 cells. Thus, a phenolic fraction with high antiproliferative activity on HL-60 cells was obtained from ASMq through the bioassay-guided fractionation process. This could provide a better pharmaceutical formulation that minimizes the administration inconveniencies of a high volume (1.5 L per day) of ASMq decoction for cancer patients.

The coumarins imperatorin and osthole are known to exert anticonvulsant activity. We have therefore analyzed the modulation of GABA-induced chloride currents (I(GABA)) by a selection of 18 coumarin derivatives on recombinant α(1)β(2)γ(2S) GABA(A) receptors expressed in Xenopus laevis oocytes by means of the two-microelectrode voltage clamp technique. Osthole (EC(50)=14 ± 1 μM) and oxypeucedanin (EC(50)=25 ± 8 μM) displayed the highest efficiency with I(GABA) potentiation of 116 ± 4 % and 547 ± 56 %, respectively. I(GABA) enhancement by osthole and oxypeucedanin was not inhibited by flumazenil (1 μM) indicating an interaction with a binding site distinct from the benzodiazepine binding site. In general, prenyl residues are essential for the positive modulatory activity, while longer side chains or bulkier residues (e.g. geranyl residues) diminish I(GABA) modulation. Generation of a binary classification tree revealed the importance of polarisability, which is sufficient to distinguish actives from inactives. A 4-point pharmacophore model based on oxypeucedanin - comprising three hydrophobic and one aromatic feature - identified 6 out of 7 actives as hits. In summary, (oxy-)prenylated coumarin derivatives from natural origin represent new GABA(A) receptor modulators.

Recent concerns about the potential carcinogenicity of estragole and methyleugenol led a number of regulatory bodies to call for restrictions on the use of herbs that contain these constituents. A number of medicinal plants produce essential oils that contain estragole and methyleugenol, including Artemisia dracunculus L. (tarragon). Previous studies have proven the antidiabetic properties of tarragon. In order to address the safety concerns of estragole containing tarragon extracts, an extraction procedure was developed to minimize the estragole and methyleugenol content in tarragon extracts and the ethanol versus aqueous extracts from two Artemisia dracunculus cultivars (French and Russian tarragon) were tested for blood glucose lowering effects in rats. It could be demonstrated that aqueous extracts of both Artemisia cultivars did not contain detectable amounts of estragole and methyleugenol, whereas ethanol extracts (60% v/v) of the French cultivar contained higher levels of the aforementioned compounds than those of the Russian cultivar. Further testing revealed that Russian tarragon lowered blood glucose levels in rats after glucose challenge, with the ethanol extract being as active as the aqueous extract. The results suggest that by using adequate production procedures the amount of potentially harmful compounds in extracts can be limited without affecting the overall pharmacological activities of these preparations.

Three new compounds, ferruginenes A (1) and B (2) and a mixture of C-5'(R) and C-5'(S) ferruginene C (3) diastereomers, have been isolated from a cytotoxic chloroform-soluble fraction of the leaves of Rhododendron ferrugineum together with 12 known compounds. The structures of these new compounds were elucidated by analyses of NMR spectroscopic and mass spectrometric data. Compounds 1-3 were tested for their cytotoxicity against three human cancer cell lines, namely, HL-60, HeLa-S3, and MCF-7.

Ethnopharmacological relevance Toad venom, called Chansu in China, has been widely used for the treatment of heart failure, sores, pains, and various cancers for a long time in clinic. Aim of the study The aim of the study is to investigate the chemical differences among a variety of toad venoms from different geographic locations and related Bufo species. Materials and methods Ten batches of commercial toad venom collected from different regions in China, one batch of fresh toad venom obtained from Bufo bufo gargarizans , and six batches of related Bufo species were analyzed by HPLC and LC-DAD-MS/MS. Individual components were identified by comparison of retention times, UV spectra, and mass spectra with authentic compounds, standard addition, as well as summarized MS fragmentation rules. Based on the profile of identified constituents and the content of cinobufagin and resibufogenin, the chemical differences observed among different samples are discussed. Results Overall, 43 compounds were identified in the methanolic extracts of the different samples of toad venom. Besides of suberoyl arginine, several free bufadienolides, bufadienolide sulfates, and suberoyl esters of bufadienolides were found. The total amounts of cinobufagin and resibufogenin, which are the only two control markers according to the current Chinese Pharmacopoeia, varied widely from 0.7% to 10.9% in the commercial Chansu samples collected in the different locations in China. Low levels of resibufogenin, but no cinobufagin was observed in the samples from Bufo melanosticus and Bufo marinus , and even neither of both compounds was found in the sample from Bufo viridis . Conclusions The chemical profiles of the different commercial and collected toad venoms from related Bufo species differed significantly, not only in the absolute and relative contents, but also in the number and type of the constituents. The main reason for this variation are species-specific differences, but additional factors, such as the harvest and post-harvest processing, and adaption to environmental factors in different geographic locations, also seem to contribute.

The concentrations of the benzoxazinoids (BAs), 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) and 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA) were determined in four selected Triticum aestivum cultivars. The impact of the cultivation period on the overall concentrations in the cultivars was determined by HPLC. Furthermore, the distribution of the two BAs was analysed in aerial and subterranean parts of the seedlings after different time periods. The highest levels were detected after 3 days. Subsequently, the levels of DIMBOA declined in all cultivars (reduction between 84% and 93%), while DIBOA was not detectable after 7 days. The distribution between aerial parts and roots varied substantially in the different cultivars. In addition, DIMBOA amounts, expressed in per cent, as well as in mg per seedling, were determined in one selected cultivar during the entire time course for better understanding of eventual dilution effects during plant growth. Our findings indicate that the consumption (up to several 100 g) of fresh sprouts (as suggested by suppliers) may already lead to exposure levels of up to 470 mg DIMBO and 190 mg of DIBOA per 100 g each. The potential adverse health effects of these doses are discussed.

The effects of cytokinins, carbohydrate sources and cold pretreatment on the conversion of protocorm-like bodies (PLBs) to shoots were investigated for the enhancement of micropropagation of Dendrobium huoshanense C.Z. Tang et S.J. Cheng, an endangered medicinal plant in China. The effects of cytokinins and carbohydrate sources on the conversion of PLBs to shoots depended on their types and concentrations. The best results in terms of shoot development from PLBs occurred on 1/2 MS medium supplemented with 20 μM kinetin and 10 g l−1 maltose. Cold pretreatment at 10 °C for 1–2 weeks significantly enhanced the conversion of PLBs to shoots, and over 1300 shoots were obtained from one gram of PLBs after 3 months of culture. The developed shoots were rooted on growth regulator-free MS medium supplemented with 8 g/l banana paste to give complete plantlets, which were successfully acclimatized with a survival rate of approximately 65%. The results indicate that a suitable cold pretreatment (10 °C for 1 week) followed by the use of 20 μM kinetin and 10 g/l maltose in 1/2 MS medium would produce a large number of shoots from PLBs for plantlet regeneration of D. huoshanense.

In some medicinal plants a specific plant-fungus association, known as arbuscular mycorrhizal (AM) symbiosis, increases the levels of secondary plant metabolites and/or plant growth. In this study, the effects of three different AM treatments on biomass and sesquiterpenic acid concentrations in two IN VITRO propagated genotypes of valerian ( VALERIANA OFFICINALIS L., Valerianaceae) were investigated. Valerenic, acetoxyvalerenic and hydroxyvalerenic acid levels were analyzed in the rhizome and in two root fractions. Two of the AM treatments significantly increased the levels of sesquiterpenic acids in the underground parts of valerian. These treatments, however, influenced the biomass of rhizomes and roots negatively. Therefore this observed increase was not accompanied by an increase in yield of sesquiterpenic acids per plant. Furthermore, one of the two genotypes had remarkably high hydroxyvalerenic acid contents and can be regarded as a hydroxyvalerenic acid chemotype.

The study was designed to investigate the hepatic metabolism and transport system of valerenic acid, a main active constituent of valerian, in isolated perfused livers from Wistar and Mrp2-deficient TR(-) rats. After administration of 20 microM valerenic acid, the formation of seven valerenic acid glucuronides (M1-M7), namely two glucuronides of valerenic acid (M6, M7), four glucuronides of hydroxylated valerenic acid (M1, M3, M4, M5), and one glucuronide of hydroxylated dehydro-valerenic acid (M2) in bile and perfusate was quantified by HPLC. The hepatic extraction ratio and clearance of valerenic acid were very high in Wistar and TR(-) rats (E: 0.983 +/- 0.006 vs. 0.981 +/- 0.004; Cl: 35.4 +/- 0.21 mL/min vs. 35.3 +/- 0.14 mL/min). However, biliary excretion and efflux of conjugates differed greatly in TR(-) rats. While cumulative biliary excretion of unconjugated valerenic acid and the glucuronides M1-M7 dropped dramatically to 1-9%, their efflux into perfusate increased 1.5- to 10-fold. This indicates that valerenic acid and its glucuronides are eliminated into bile by Mrp2. In summary, valerenic acid was metabolized to several conjugates, whereby the canalicular transporter Mrp2 mediated biliary excretion of the parent drug and its glucuronides.

Aconitum species have been used in China as an essential drug in Traditional Chinese Medicine (TCM) for 2000 years. Reviewing the clinical application of Aconitum, their pharmacological effects, toxicity and detoxifying measures, herb-herb interactions, clinical taboos, famous herbal formulas, traditional and current herbal processing methods based upon a wide range of literature investigations serve as a case study to explore the multidisciplinary implications of botanicals used in TCM. The toxicological risk of improper usage of Aconitum remains very high, especially in countries like China, India and Japan. The toxicity of Aconitum mainly derives from the diester diterpene alkaloids (DDAs) including aconitine (AC), mesaconitine (MA) and hypaconitine (HA). They can be decomposed into less or non-toxic derivatives through Chinese traditional processing methods (Paozhi), which play an essential role in detoxification. Using Paozhi, the three main forms of processed aconite -- yanfuzi, heishunpian and baifupian -- can be obtained (CPCommission, 2005). Moreover, some new processing techniques have been developed in China such as pressure-steaming. The current development of fingerprint assays, in particular HPLC, has set a good basis to conduct an appropriate quality control for TCM crude herbs and their ready-made products. Therefore, a stipulation for a maximum level of DDA content of Aconitum is highly desirable in order to guarantee the clinical safety and its low toxicity in decoctions. Newly developed HPLC methods have made the accurate and simultaneous determination and quantification of DDA content interesting.

Objectives Cimicifuga racemosa (Actaea racemosa, black cohosh) is used as an anti-inflammatory, antipyretic and analgesic remedy in traditional medicines. The present study focuses on the effects of C. racemosa root extracts on inducible nitric oxide synthase (iNOS) in lipopolysaccharide-stimulated murine macrophages (RAW 264.7). Methods C. racemosa rhizome and phosphate-buffered saline extracts were analysed for phenolcarboxylic acids and triterpene glycosides using an HPLC photodiode array/evaporative light-scattering detector system. iNOS was characterised by measurement of iNOS protein (immunoblotting), iNOS mRNA (semiquantitative competitive RT-PCR), nitric oxide production (nitrite levels) and nuclear translocation of nuclear factor-kB (p65 subunit) protein. Key findings Incubation of lipopolysaccharide-stimulated macrophages with aqueous C. racemosa extracts (0–6 mg/ml) inhibited nitrite accumulation in a concentration-dependent manner. C. racemosa extracts also reduced iNOS protein expression and iNOS mRNA levels in a dose-dependent manner. C. racemosa extracts did not significantly inhibit iNOS activity and did not affect nuclear translocation of nuclear factor-kB (p65 subunit) protein. Incubation with the extract was associated with a concentration-dependent reduction of interferon beta and interferon regulatory factor 1 mRNA. Among the triterpene glycosides, 23-epi-26-deoxyactein was identified as an active principle in C. racemosa extracts. Conclusions Extracts from the roots of C. racemosa inhibit nitric oxide production by reducing iNOS expression without affecting activity of the enzyme. This might contribute to the anti-inflammatory activities of C. racemosa.

Valeriana Officinalis L . is a traditionally used sleep remedy, however, the mechanism of action and the substances responsible for its sedative and sleep-enhancing properties are not fully understood. As we previously identified valerenic acid as a subunit-specific allosteric modulator of GABAA receptors, we now investigated the relation between modulation of GABAA receptors by Valerian extracts of different polarity and the content of sesquiterpenic acids (valerenic acid, acetoxyvalerenic acid). All extracts were analysed by HPLC concerning the content of sesquiterpenic acids. GABAA receptors composed of alpha 1, beta 2 and gamma 2S subunits were expressed in Xenopus laevis oocytes and the modulation of chloride currents through GABAA receptors (IGABA) by Valerian extracts was investigated using the two-microelectrode voltage clamp technique. Apolar extracts induced a significant enhancement of IGABA, whereas polar extracts showed no effect. These results were confirmed by fractionating a highly active ethyl acetate extract: again fractions with high contents of valerenic acid exhibited strong receptor activation. In addition, removal of sesquiterpenic acids from the ethyl acetate extract led to a loss of I (GABA) enhancement. In conclusion, our data show that the extent of GABAA receptor modulation by Valerian extracts is related to the content of valerenic acid.

In our chemotaxonomic investigation of pharmaceutically relevant Crataegus species, the qualitative and quantitative flavonoid fingerprint of Crataegus monogyna and C. pentagyna is presented. Six flavonoids were identified as vitexin-2''-O-rhamnoside (1), vitexin (2), isovitexin (3), rutin (4), hyperoside (5), and isoquercitrin (6). Besides the verification of the main compounds isoorientin (7) and orientin (8) in C. pentagyna, further four flavonoids were isolated and identified as isoorientin-2''-O-rhamnoside (9), orientin-2''-O-rhamnoside (10), isovitexin-2''-O-rhamnoside (11), and 8-methoxykaempferol-3-O-glucoside (12) by means of 1D- and 2D-NMR, MS, and UV analyses. Compound 12 was isolated for the first time from C. pentagyna. In contrast to C. pentagyna, C. monogyna samples were predominated by 4'''-acetylvitexin-2''-O-rhamnoside (13), which was missing in C. pentagyna. Hence, 13 represents an interesting compound for chemotaxonomy of C. monogyna, whereas the main flavonoids 7, 8, and 12 could be proposed as markers for C. pentagyna. The absence of 7, 8, 12, and 13 in C. laevigata offers an appropriate tool for additional differentiation from C. monogyna and C. pentagyna, and for sample identification and quality control of the three main Crataegus species used in European phytotherapy.

The so-called hand-foot syndrome (HFS) is a dose-limiting side effect with only rare therapeutic options in cancer patients treated with capecitabine (Xeloda). Depending on the intensity of the skin reaction dose reduction, interruption or even the break-off of capecitabine therapy is necessary. We therefore try to describe a new and promising alternative treatment of the hand-foot syndrome, a local therapy with a mixture of herbal medicinal products mainly consisting of hand- and foot baths, and present the promising results of this treatment modality observed in 11 consecutive patients.

Achillea millefolium L. s.l. is a cytogenetically, morphologically, and chemically polymorphic aggregate. Besides the sesquiterpenes that possess chemotaxonomic relevance and mediate the antiphlogistic activity, the plant contains phenolic compounds such as dicaffeoylquinic acids and flavonoids causing choleretic and spasmolytic effects. To evaluate their contribution to the chemotaxonomy of European taxa of the A. millefolium group, we developed a SPE-HPLC/UV method that allows quantification of the phenolic constituents in the different taxa. The investigated species displayed differences in the quantitative and qualitative composition of phenolic acids and flavonoids. Hence, they seem to be of chemotaxonomic significance, especially for the distinction of the diploid taxa. Combining the obtained results with the data of the sesquiterpene analyses gives a comprehensive insight into the distribution of those pharmacologically relevant plant constituents in the A. millefolium group.

Benzoxazinoids (BAs) are toxic constituents of sprouts of Gramineae such as wheat, maize and rye and are part of the plant defence system against pests. In the last years, sprouts have been increasingly consumed as health foods and are also used for the production of dietary supplements. In the present study we investigated the mutagenic activities of the two most abundant BAs, namely 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) and 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA) in the Salmonella/microsome assay and additionally, in micronucleus (MN) assay and single cell gel electrophoresis (SCGE) assay in a human-derived liver cell line (HepG2). DIBOA caused significant induction of his(+) revertants in all three strains in the range between 0.02 and 0.50 mg/plate; the highest activity was observed in TA100 (fivefold increase over the background at the highest dose level). The effect in YG1024 (a derivative of TA98 with increased acetyltransferase activity) was only slightly higher than the effect in the parental strain indicating that acetylation plays no crucial role in the activation of this BA. DIMBOA was in general less active and a positive result was only seen in the base substitution strain (TA100). Addition of rat liver homogenate (S9-mix) led to a significant (ca. twofold) increase of the mutagenic activities of both BAs. In SCGE assays with HepG2 cells consistently negative results were obtained with both compounds whereas in MN assays significant dose dependent effects were observed under similar experimental conditions. DIMBOA caused significant effects already at concentrations > or =1 microM; at the highest dose (20 microM) the MN frequency was sevenfold higher than the background level. DIBOA caused weaker effects and was positive at doses > or =2.5 microM, the maximal induction (twofold over background) was observed with 20 microM. Overall, DIMBOA was ca. 30-fold more active as DIBOA. Subsequent experiments with pancentromeric probes showed that >80% of the MN induced at the highest doses gave a centromere positive signal indicating that both BAs are aneugenic. This is an interesting observation as it is assumed that aneuploidy is a key event in cancer induction and at present no other aneugenic plant-derived substances of dietary relevance are known.

The major effects of arbuscular mycorrhizal fungi (AMF) colonization on plant growth, monoterpenoids and coumarins were determined in two genotypes of angelica (Angelica archangelica L., Apiaceae). Genetically uniform experimental plants were used, which was achieved by in vitro propagation of two genotypes (individual plants). The effects of Glomus mosseae (Funneliformis mosseae, BEG 12), Glomus intraradices (Rhizophagus intraradices, BB-E) and the AMF mixture Symbivit® were tested against a negative (without AMF and without additional phosphorus) and a positive (KH2PO4 without AMF) control in five plants per treatment. All in all 50 plants were investigated. Fifteen monoterpenoid and seven coumarin compounds were quantitatively determined in the rhizome and coarse root fractions by means of GC/MS using an internal standard. The sum of the monoterpenoids and coumarins was calculated. Concentrations of compounds rather slightly decreased upon inoculation with AMF when compared to the control. In contrast, biomass increased showing the highest amounts for plants treated with G. mosseae. These results finally caused a marked increase in yield for G. mosseae treated plants compared to the control. Phosphorus treatment led to the lowest yield with significant results for coumarins and in particular for osthole compared to G. mosseae treatment. This is also evidence of an independent effect from an increased phosphorus uptake due to root colonization by AMF. In summary, the results showed a marked increase in yield of all investigated compounds through treatment with G. mosseae compared to the control group, even if the level of significance was just barely missed presumably on account of the small sample size.

Medicinal plants have historically proven their value as a source of molecules with therapeutic potential, and nowadays still represent an important pool for the identification of novel drug leads. In the past decades, pharmaceutical industry focused mainly on libraries of synthetic compounds as drug discovery source. They are comparably easy to produce and resupply, and demonstrate good compatibility with established high throughput screening (HTS) platforms. However, at the same time there has been a declining trend in the number of new drugs reaching the market, raising renewed scientific interest in drug discovery from natural sources, despite of its known challenges. In this survey, a brief outline of historical development is provided together with a comprehensive overview of used approaches and recent developments relevant to plant-derived natural product drug discovery. Associated challenges and major strengths of natural product-based drug discovery are critically discussed. A snapshot of the advanced plant-derived natural products that are currently in actively recruiting clinical trials is also presented. Importantly, the transition of a natural compound from a “screening hit” through a “drug lead” to a “marketed drug” is associated with increasingly challenging demands for compound amount, which often cannot be met by re-isolation from the respective plant sources. In this regard, existing alternatives for resupply are also discussed, including different biotechnology approaches and total organic synthesis. While the intrinsic complexity of natural product-based drug discovery necessitates highly integrated interdisciplinary approaches, the reviewed scientific developments, recent technological advances, and research trends clearly indicate that natural products will be among the most important sources of new drugs also in the future.

In addition to the importance of many Dioscorea species (yams) as starchy staple food, some representatives are known and still used as a source for the steroidal sapogenin diosgenin, which, besides phytosterols derived from tall-oil, is an important precursor for partial synthesis of steroids for pharmaceutical research and applications. While in edible yams the diosgenin content should be as low as possible, a high yield of the compound is preferable for cultivars which are grown for the extraction of sterols. In the past, miscalculations and insufficiently precise techniques for quantification of diosgenin prevailed. Therefore we set out to re-evaluate the steroid content of a world collection of Dioscorea species, using leaves as sample material. We optimized diosgenin quantification techniques and fingerprinted the whole collection with the DNA amplification fingerprinting (DAF) technique. Total diosgenin contents ranged from 0.04 to 0.93% of dry weight within the collection. Several Dioscorea cultivars can be characterized via their DAF fingerprint patterns.

A micropropagation protocol for squill (Charybdis numidica, Hyacinthaceae) was developed using nodule culture. Nodule formation on leaf sections was induced in liquid Murashige and Skoog (MS) medium supplemented with 20 microM N6-benzylaminopurine (BA) under dark conditions. Nodules were cultured on semi-solid MS medium with factorial combinations of BA (0-40 microM) and alpha-naphthaleneacetic acid (NAA) (0-10 microM) under continuous light. Shoot regeneration from nodules occurred at varying degrees on all media. The highest number of shoots was formed on medium containing 2.5 microM NAA and 20 microM BA, while the maximum number of regenerated bulblets per gram nodule was induced on culture medium supplemented with 2.5 microM NAA alone. Regenerated shoots were successfully rooted at approximately 92% on semi-solid MS medium supplemented with 10 microM indole-3-acetic acid (IAA). Plantlets could be hardened and grew well after transfer to the greenhouse. Chemical analyses showed consistent bufadienolide patterns from cloned plantlets and the mother plant.

Bauhinia variegate L. is a multipurpose tree and its micropropagation holds great promise in agroforestry. The sterilized seeds were first inoculated on Murashige and Skoog (MS) medium. Nodal cuttings from these seedlings grown in vitro were used as explants for micropropagation. Nodal cutting inoculated on the medium with various concentrations of BAP (benzyl-aminopurine) and NAA (£- naphthalene acetic acid) and separately generated varied results. Best propagation was recorded on MS medium supplemented with 1.0 ?M BAP with 0.05 ?M NAA. Propagated plants were successfully acclimatized and rooted in pots (6 cm diameter) containing soil and sand in1:1 ratio and then finally transferred to the field. All the data generated were analysed statistically using SPSS statistical package. Nepal Journal of Science and Technology Vol. 13, No. 1 (2012) 39-41 DOI: http://dx.doi.org/10.3126/njst.v13i1.7397

Cardiac glycosides are commonly used drugs in clinical medicine. We analyzed the cytotoxic effect of six steroids belonging to the bufadienolide family on malignant T lymphoblasts and normal peripheral blood mononuclear cells (PBMC). One compound was a natural bufadienolide glycoside (hellebrin) with cardiac activity. The other five compounds were chemically modified derivatives that did not contain cardioactive groups. We found that these steroids were able to cause time-dependent apoptosis in Jurkat T lymphoblasts, whereas they only minimally affected PBMC. Preferential killing of malignant cells was induced by the natural cardioactive substance hellebrin and by three of the five chemically modified non-cardioactive derivatives. The substances caused mitochondrial transmembrane potential disruption and internucleosomal DNA fragmentation in tumor cells. The cytoplasmic and nuclear events of bufadienolide-induced apoptosis were strongly inhibited in the presence of caspase 8, caspase 9, or caspase 3 inhibitors, as well as in the presence of the broad-spectrum caspase inhibitor Z-VAD-FMK. Overexpression of Bcl-2 significantly protected bufadienolide-treated cells from phosphatidylserine translocation, transmembrane potential disruption, and internucleosomal DNA fragmentation. Our results show that the analyzed bufadienolide derivatives preferentially kill malignant human lymphoblasts by initiating apoptosis via the classical caspase-dependent pathway. Apoptosis-inducing agents specific for tumor cells might be ideal anti-tumor drugs. The therapeutic use of bufadienolides has been hampered by their concomitant cardiac activity. The description of compounds without cardiac activity but with tumor-specific cytotoxicity suggests the potential of using them in cancer therapy.

A simple method for the rapid separation and quantification of flavone-O- and C-glycosides in A. setacea W. et K. by capillary zone electrophoresis (CZE) with UV detection is described. Using 25 mM sodium borate with 20% (v/v) of methanol (pH 9.3) as running buffer sufficient separation of the analytes was achieved within 19 min. For the quantitative determination isorhamnetin-3-O-rutinoside was used as internal standard. The method was successfully applied to a rapid characterisation of the flavonoid complex and a precise quantification of the single and total amount of the flavonoids in different samples of A. setacea.

Capillary electrophoresis has been applied for the separation and quantification of the flavonoids in Passiflorae herba. Separations were performed using 25 mM sodium borate with 20 % methanol (pH 9.5). For the quantification quercetin 3-O-arabinoside was used as internal standard. The method was applied to the determination of the flavonoid glycosides in 10 different commercial samples of the drug and showed similar flavonoid patterns, but differences concerning the single and total amounts of flavonoids. The total flavonoid contents determined with the new method correlated satisfactorily with those achieved by the spectrophotometric assay according to the European Pharmacopoeia.

The detailed investigation of a methanolic extract of aerial parts of Achillea nobilis resulted in the isolation of 10 flavonoids. A new C-glycosylflavone, luteolin-6-C-apiofuranosyl-(1‴→2″)-glucoside, was isolated besides orientin, isoorientin, vitexin, isoschaftoside, luteolin- 7-O-β-glucuronide, luteolin-4′-O-β-glucoside and quercetin-3-O-methyl ether and two rare flavonolglycosides, quercetin-3-O-α-arabinosyl-(1‴→6″ )-glucoside and quercetin-3-O-methylether-7-O-β-glucoside. The structures were established either by comparison with authentic substances or by UV, 1H NMR and 13C NMR spectroscopic methods including 2ᴅ-NMR techniques and ESI-MS

Pluchea odorata is ethno pharmaceutically used to treat inflammation-associated disorders. The dichloromethane extract (DME) was tested in the carrageenan-induced rat paw oedema assay investigating its effect on inflammation that was inhibited by 37%. Also an in vitro anti-neoplastic potential was reported. However, rather limited information about the bio-activity of purified compounds and their cellular mechanisms are available. Therefore, two of the most abundant eudesmanes in P. odorata were isolated and their anti-neoplastic and anti-intravasative activities were studied. HL-60 cells were treated with P. odorata compounds and metabolic activity, cell number reduction, cell cycle progression and apoptosis induction were correlated with relevant protein expression. Tumour cell intravasation through lymph endothelial monolayers was measured and potential causal mechanisms were analyzed by Western blotting. Compound PO-1 decreased the metabolic activity of HL-60 cells (IC 50 = 8.9 μM after 72 h) and 10 μM PO-1 induced apoptosis, while PO-2 showed just weak anti-neoplastic activities at concentrations beyond 100 μM. PO-1 arrested the cell cycle in G1 and this correlated with induction of JunB expression. Independent of this mechanism 25 μM PO-1 decreased MCF-7 spheroid intravasation through the lymph endothelial barrier. Hence, PO-1 inhibits an early step of metastasis, impairs unrestricted proliferation and induces apoptosis at low micromolar concentrations. These results warrant further testing in vivo to challenge the potential of PO-1 as novel lead compound.

Cardiotonic steroids have long been in clinical use for treatment of heart failure and are now emerging as promising agents in various diseases, especially cancer. Their main target is Na(+)/K(+)-ATPase, a membrane protein involved in cellular ion homeostasis. Na(+)/K(+)-ATPase has been implicated in cancer biology by affecting several cellular events and signaling pathways in both sensitive and drug-resistant cancer cells. Hence, we investigated the cytotoxic activities of 66 cardiotonic steroids and cardiotonic steroid derivatives in sensitive CCRF-CEM and multidrug-resistant CEM/ADR5000 leukemia cells. Data were then subjected to quantitative structure-activity relationship analysis (QSAR) and molecular docking into Na(+)/K(+)-ATPase, which both indicated a possible differential expression of the pump in the mentioned cell lines. This finding was confirmed by western blotting, intracellular potassium labeling and next generation sequencing which showed that Na(+)/K(+)-ATPase was less expressed in multidrug-resistant than in sensitive cells.

An apolar extract of the traditional medicinal plant Neurolaena lobata inhibited the expression of the NPM/ALK chimera, which is causal for the majority of anaplastic large cell lymphomas (ALCLs). Therefore, an active principle of the extract, the furanoheliangolide sesquiterpene lactone lobatin B, was isolated and tested regarding the inhibition of ALCL expansion and tumour cell intravasation through the lymphendothelium. ALCL cell lines, HL-60 cells and PBMCs were treated with plant compounds and the ALK inhibitor TAE-684 to measure mitochondrial activity, proliferation and cell cycle progression and to correlate the results with protein- and mRNA-expression of selected gene products. Several endpoints indicative for cell death were analysed after lobatin B treatment. Tumour cell intravasation through lymphendothelial monolayers was measured and potential causal mechanisms were investigated analysing NF-κB- and cytochrome P450 activity, and 12(S)-HETE production. Lobatin B inhibited the expression of NPM/ALK, JunB and PDGF-Rβ, and attenuated proliferation of ALCL cells by arresting them in late M phase. Mitochondrial activity remained largely unaffected upon lobatin B treatment. Nevertheless, caspase 3 became activated in ALCL cells. Also HL-60 cell proliferation was attenuated whereas PBMCs of healthy donors were not affected by lobatin B. Additionally, tumour cell intravasation, which partly depends on NF-κB, was significantly suppressed by lobatin B most likely due to its NF-κB-inhibitory property. Lobatin B, which was isolated from a plant used in ethnomedicine, targets malignant cells by at least two properties: I) inhibition of NPM/ALK, thereby providing high specificity in combating this most prevalent fusion protein occurring in ALCL; II) inhibition of NF-κB, thereby not affecting normal cells with low constitutive NF-κB activity. This property also inhibits tumour cell intravasation into the lymphatic system and may provide an option to manage this early step of metastatic progression.