Isolation and NMR Scaling Factors for the Structure Determination of Lobatolide H, a Flexible Sesquiterpene from Neurolaena lobata.

A new flexible germacranolide (1, lobatolide H) was isolated from the aerial parts of Neurolaena lobata. The structure elucidation was performed by classical NMR experiments and DFT NMR calculations. Altogether, 80 theoretical level combinations with existing 13C NMR scaling factors were tested, and the best performing ones were applied on 1. 1H and 13C NMR scaling factors were also developed for two combinations utilizing known exomethylene containing derivatives, and the results were complemented by homonuclear coupling constant (JHH) and TDDFT-ECD calculations to elucidate the stereochemistry of 1. Lobatolide H possessed remarkable antiproliferative activity against human cervical tumor cell lines with different HPV status (SiHa and C33A), induced cell cycle disturbance and exhibited a substantial antimigratory effect in SiHa cells.

Isolation, Structure Determination of Sesquiterpenes from Neurolaena lobata and Their Antiproliferative, Cell Cycle Arrest-Inducing and Anti-Invasive Properties against Human Cervical Tumor Cells.

Seven new germacranolides (1-3, 5-8), among them a heterodimer (7), and known germacranolide (4), eudesmane (9) and isodaucane (10) sesquiterpenes were isolated from the aerial parts of Neurolaena lobata. Their structures were determined by using a combination of different spectroscopic methods, including HR-ESIMS and 1D and 2D NMR techniques supported by DFT-NMR calculations. The enantiomeric purity of the new compounds was investigated by chiral HPLC analysis, while their absolute configurations were determined by TDDFT-ECD and OR calculations. Due to the conformationally flexible macrocycles and difficulties in assigning the relative configuration, 13C and 1H NMR chemical shift and ECD and OR calculations were performed on several stereoisomers of two derivatives. The isolated compounds (1-10) were shown to have noteworthy antiproliferative activities against three human cervical tumor cell line with different HPV status (HeLa, SiHa and C33A). Additionally, lobatolide C (6) exhibited substantial antiproliferative properties, antimigratory effect, and it induced cell cycle disturbance in SiHa cells.

Flavonoid, stilbene and diarylheptanoid constituents of Persicaria maculosa Gray and cytotoxic activity of the isolated compounds.

Persicaria maculosa (Polygonaceae) has been used as edible and as medicinal plant since ancient times. As a result of multistep chromatographic purifications, chalcones [2'-hydroxy-3',4',6'-trimethoxychalcone (1), pashanone (2), pinostrobin chalcone (3)], flavanones [6-hydroxy-5,7-dimethoxyflavanone (4), pinostrobin (5), onysilin (6), 5-hydroxy-7,8-dimethoxyflavanone (7)], flavonol [3-O-methylgalangin (8)], stilbene [persilben (9)], diarylheptanoids [1,7-diphenylhept-4-en-3-one (10), dihydroyashabushiketol (12), yashabushidiol B (13)] and 3-oxo-α-ionol-glucoside (11) were isolated from P. maculosa. The present paper reports for the first time the occurrence of diarylheptanoid-type constituents in the family Polygonaceae. Cytotoxicity of 1-5, 7 and 9-11 on 4 T1 mouse triple negative breast cancer cells was assayed by MTT test. None of the tested compounds reduced the cell viability to less than 80% of the control. On non-tumorigenic D3 human brain endothelial cells the decrease of cell viability was observed in case of 1 and 2. Further impedance measurements on 4 T1 and D3 cells a concentration-dependent decrease in the cell index of both cell types was demonstrated for 1, while 2 proved to be toxic only on endothelial cells.

Inhibition of COX-2 and NF-κB1 Gene Expression, NO Production, 5-LOX, and COX-1 and COX-2 Enzymes by Extracts and Constituents of Onopordum acanthium.

The present study focused on the investigation of the inhibition of cyclooxygenase-2 and nuclear factor kappa B1 gene expression, nitric oxide production, leukotriene biosynthesis (5-lipoxygenase), and cyclooxygenase-1 and cyclooxygenase-2 enzymes of Onopordum acanthium, and the isolation and identification of its active compounds. From the chloroform soluble part of the MeOH extract prepared from aerial parts, lignans [pinoresinol (1), syringaresinol (2), and medioresinol (3)] and flavonoids [hispidulin (4), nepetin (5), apigenin (6), and luteolin (7)] were isolated by a combination of different chromatographic methods. The structures of the compounds were determined by means of mass spectrometry and 1D- and 2D-nuclear magnetic resonance spectroscopy, and by comparison of the spectral data with literature values. Extracts of different polarity and the isolated compounds obtained from the aerial parts, together with those previously isolated from the roots of the plant [4ß,15-dihydro-3-dehydrozaluzanin C (8), zaluzanin C (9), 4ß,15,11ß,13-tetrahydrozaluzanin C (10), nitidanin diisovalerianate (11), 24-methylenecholesterol (12), and 13-oxo-9Z,11E-octadecadienoic acid (13)], were evaluated for their inhibitory effects on cyclooxygenase-2 and nuclear factor kappa B1 gene expression, inducible nitric oxide synthase, 5-lipoxygenase, and cyclooxygenase-1 and cyclooxygenase-2 enzymes in in vitro assays. It was found that O. acanthium extracts exert strong inhibitory activities in vitro and some lignans, flavonoids, and sesquiterpenes may play a role in these activities. 4ß,15-Dihydro-3-dehydrozaluzanin C and zaluzanin C at 20 µM were the most active constituents tested against lipopolysaccharide/interferon-γ-induced nitric oxide production (100.4 ± 0.5 % and 99.4 ± 0.8 %) in the inhibition of cyclooxygenase-2 (98.6 ± 0.2 % and 97.0 ± 1.1 %) and nuclear factor kappa B1 gene expression (76.7 ± 7.3 % and 69.9 ± 3.4 %). Furthermore, it was shown that these inhibitory effects are not due to cytotoxicity of the compounds.

The germacranolide sesquiterpene lactone neurolenin B of the medicinal plant Neurolaena lobata (L.) R.Br. ex Cass inhibits NPM/ALK-driven cell expansion and NF-κB-driven tumour intravasation.

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-Rß 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.

Lobatin B inhibits NPM/ALK and NF-κB attenuating anaplastic-large-cell-lymphomagenesis and lymphendothelial tumour intravasation.

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-Rß, 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.

Xanthine oxidase inhibitory activity of extracts prepared from Polygonaceae species.

The xanthine oxidase (XO) inhibitory activity of aqueous and organic extracts of 27 selected species belonging in five genera (Fallopia, Oxyria, Persicaria, Polygonum and Rumex) of the family Polygonaceae occurring in the Carpathian Basin were tested in vitro. From different plant parts (aerial parts, leaves, flowers, fruits and roots), a total of 196 extracts were prepared by subsequent extraction with methanol and hot H2O and solvent-solvent partition of the MeOH extract yielding n-hexane, chloroform and 50% MeOH subextracts. It was found that the chloroform subextracts and/or the remaining 50% MeOH extracts of Fallopia species (F. bohemica, F. japonica and F. sachalinensis), Rumex species (R. acetosa, R. acetosella, R. alpinus, R. conglomeratus, R. crispus, R. hydrolapathus, R. pulcher, R. stenophyllus, R. thyrsiflorus, R. obtusifolius subsp. subalpinus, R. patientia) and Polygonum bistorta, Polygonum hydropiper, Polygonum lapathifolium and Polygonum viviparum demonstrated the highest XO inhibitory activity (>85% inhibition) at 400 µg/mL. The IC50 values of the active extracts were also determined. On the basis of the results, these plants, and especially P. hydropiper and R. acetosella, are considered worthy of activity-guided phytochemical investigations.

Sesquiterpenes from Neurolaena lobata and their antiproliferative and anti-inflammatory activities.

Five new sesquiterpenes, neurolobatin A (1), neurolobatin B (2), 5ß-hydroxy-8ß-isovaleroyloxy-9α-hydroxycalyculatolide (3), 3-epi-desacetylisovaleroylheliangine (4), and 3ß-acetoxy-8ß-isovaleroyloxyreynosin (5), were isolated from the aerial parts of Neurolaena lobata. The structures were established by means of a combined spectroscopic data analysis, including ESIMS, APCI-MS, and 1D- and 2D-NMR techniques. Neurolobatin A (1) and B (2) are unusual isomeric seco-germacranolide sesquiterpenes with a bicyclic acetal moiety, compounds 3 and 4 are unsaturated epoxy-germacranolide esters, and compound 5 is the first eudesmanolide isolated from the genus Neurolaena. The isolated compounds (1-5) were shown to have noteworthy antiproliferative activities against human tumor cell lines (A2780, A431, HeLa, and MCF7). The anti-inflammatory effects of 1-5, evaluated in vitro using LPS- and TNF-α-induced IL-8 expression inhibitory assays, revealed that all these compounds strongly down-regulated the LPS-induced production of IL-8 protein, with neurolobatin B (2) and 3-epi-desacetylisovaleroylheliangine (4) being the most effective.

Inhibition of G protein-activated inwardly rectifying K+ channels by extracts of Polygonum persicaria and isolation of new flavonoids from the chloroform extract of the herb.

The G protein-activated inwardly rectifying K+ channel-modulatory activities of Polygonum persicaria extracts were investigated by using an automated patch-clamp method, with the aim of identifying natural sources of promising ion channel-blocking compounds. The chloroform extract of the whole plant at 0.1 mg/mL exhibited high G protein-activated inwardly rectifying K+ channel-inhibitory activity. Fractionation of this extract by vacuum liquid chromatography on RP-silica gel resulted in 6 fractions, which were evaluated for G protein-activated inwardly rectifying K+ channel-modulatory activity. RP-HPLC of the most active fractions afforded the main compounds 1-4 in pure form and a mixture containing the minor constituents. The structures were identified by means of UV, HRMS, and advanced NMR methods as 3-O-senecioyl-isorhamnetin (1), 3-O-angeloyl-isorhamnetin (2), 5,3',4',5'-tetramethoxy-6,7-methylenedioxyflavone (3), and 3,5,3',4',5'-pentamethoxy-6,7-methylenedioxyflavone (4). Compounds 1-4 are new natural products, though 4 was reported earlier as a synthetic compound. Neither the individual, nor the combined application of compounds 1-4 modified the G protein-activated inwardly rectifying K+ channel activity. However, a marked G protein-activated inwardly rectifying K+ current-inhibitory effect was detected on use of the HPLC eluates containing the minor compounds. These results indicate the presence of electrophysiologically active agents among the minor compounds.

Antiproliferative activity of polygonaceae species from the Carpathian Basin against human cancer cell lines.

Aqueous and organic extracts of 27 selected species from five genera (Fallopia, Oxyria, Persicaria, Polygonum and Rumex) of the family Polygonaceae occurring in the Carpathian Basin were screened in vitro for antiproliferative activity against HeLa (cervix epithelial adenocarcinoma), A431 (skin epidermoid carcinoma) and MCF7 (breast epithelial adenocarcinoma) cells, using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. A total of 196 n-hexane, chloroform, 50% methanol or water extracts of different plant parts were investigated. It was found that extracts of Polygonum hydropiper, Rumex acetosa, Rumex alpinus, Rumex aquaticus, Rumex scutatus and Rumex thyrsiflorus at 10 or 30 µg/mL demonstrated substantial cell growth inhibitory activity (at least 50% inhibition of cell proliferation) against one or more cell lines. R. acetosa and R. thyrsiflorus proved to be the most active and are considered worthy of activity-guided phytochemical investigations.