Pauciflorins A-E, Unexpected Chromone-Monoterpene-Derived Meroterpenoids from Centrapalus pauciflorus.

Five unusual meroterpenoids based on new carbon skeletons, pauciflorins A-E (1-5), were isolated by multistep chromatographic separations of a methanol extract of the aerial parts of Centrapalus pauciflorus. Compounds 1-3 are derived by the connection of a 2-nor-chromone and a monoterpene unit, whereas 4 and 5 are dihydrochromone-monoterpene adducts with a rarely occurring orthoester functionality. The structures were solved using 1D and 2D NMR, HRESIMS, and single-crystal X-ray diffraction. Pauciflorins A-E were evaluated for antiproliferative activity against human gynecological cancer cell lines, but were inactive (IC50 < 10 µM) in each case.

12-Deoxyphorbol Esters Induce Growth Arrest and Apoptosis in Human Lung Cancer A549 Cells Via Activation of PKC-δ/PKD/ERK Signaling Pathway.

Prostratin, a non-tumor promoting 12-deoxyphorbol ester, has been reported as a protein kinase C (PKC) activator and is shown to have anti-proliferative activity in certain cancer cell types. Here we show that GRC-2, a prostratin analogue isolated from Euphorbia grandicornis, is ten-fold more potent than prostratin for inhibiting the growth of human non-small cell lung cancer (NSCLC) A549 cells. Flow cytometry assay revealed that GRC-2 and prostratin inhibited cell cycle progression at the G2/M phase and induced apoptosis. The cytotoxic effect of GRC-2 and prostratin was accompanied by activation and nuclear translocation of PKC-δ and PKD as well as hyperactivation of extracellular signal-related kinase (ERK). Knockdown of either PKC-δ, PKD or ERK significantly protected A549 cancer cells from GRC-2- and prostratin-induced growth arrest as well as apoptosis. Taken together, our results have shown that prostratin and a more potent analogue GRC-2 reduce cell viability in NSCLC A549 cells, at least in part, through activation of the PKC-δ/PKD/ERK pathway, suggesting the potential of prostratin and GRC-2 as anticancer agents.

Diterpenoids from Euphorbia dulcis with Potassium Ion Channel Inhibitory Activity with Selective G Protein-Activated Inwardly Rectifying Ion Channel (GIRK) Blocking Effect.

Nine new (1-9) and two known (10, 11) jatrophane diterpenoids were isolated from the methanol extract of Euphorbia dulcis. The structure elucidation of the compounds was performed by means of extensive spectroscopic analysis, including HRESIMS, 1D (1H, JMOD), and 2D (HSQC, HMBC, 1H-1H-COSY, NOESY) NMR experiments. The absolute configuration of compound 1 was determined by single-crystal X-ray diffraction. The electrophysiological effects of compounds 1-11 and the five diterpenoids (12-16) previously isolated from Euphorbia taurinensis were investigated on stable transfected HEK-GIRK1/4 (Kir3.1/3.4) and HEK-hERG (Kv11.1) cell lines using automated patch-clamp equipment. The majority of the diterpenoids showed significant blocking activity on GIRK channels (60.8-88.7% at 10 µM), while compounds 1, 2, 9-11, 13, and 14 exerted notable inhibitory effects even at 1 µM concentration. None of the jatrophane diterpenoids interfered with the function of hERG proteins; however, compound 14 remarkably hampered K+ flow through hERG channels. These selective activities suggest that jatrophane diterpenoids may represent a group of potential lead compounds for the development of novel therapeutic agents against atrial fibrillation.

Bioactivity-Guided Investigation of the Anti-Inflammatory Activity of Hippophae rhamnoides Fruits.

According to modern ethnobotanical records, the fruit of Hippophae rhamnoides is effective in the treatment of different allergic symptoms. In order to obtain pharmacological evidence for this observation, the fruit was investigated for anti-inflammatory activity using in vivo animal models. Aqueous and 70% MeOH extracts were tested in 48/80-induced rat paw edema assay after oral administration, and it was found that the 70% MeOH extract (500 mg/kg) reduced significantly edema volume (0.660 ± 0.082 mL vs. control 0.935 ± 0.041 mL). Extracts of different parts of the fruit (pulp, peel, seed) were investigated in the same assay, and the peel extract was shown to exhibit maximum edema-reducing effect (0.470 ± 0.124 mL vs. control 0.920 ± 0.111 mL). This extract was used to elucidate the mode of action. Different inflammation inducers (serotonin, histamine, dextran, bradykinin, and carrageenan) were applied in the rat paw model, but the extract inhibited only the compound 48/80 elicited inflammation. The active extract was then fractionated by solvent-solvent partitioning and chromatographic methods with the guidance of the 48/80-induced anti-inflammatory assay, and the main compounds responsible for the activity were identified as ursolic acid and oleanolic acid. Our data suggest that the activity is most probably based on a membrane stabilizing effect caused by the inhibition of degranulation of mast cells. Moreover, previously unknown 2,5-bis-aryl-3,4-dimethyltetrahydrofuran lignans, nectandrin B, fragransin A2, and saucernetindiol were isolated and identified from H. rhamnoides for the first time.

Bioactive Segetane, Ingenane, and Jatrophane Diterpenes from Euphorbia taurinensis.

A novel segetane (1: ) and jatrophane diterpene (2: ), together with five known diterpenoids possessing segetane (3: ), jatrophane (4: ), and ingenane skeletons (5 - 7: ), were isolated from the methanol extract of Euphorbia taurinensis All. The structure elucidation of the compounds was performed by means of extensive spectroscopic analysis, including HRESIMS and 1D (1H, J-modulated spin-echo carbon experiment) and 2D (HSQC, HMBC, COSY, NOESY) NMR experiments. The multidrug resistance reversing and cytotoxic effects of five diterpenes (1, 4:  - 7: ) were studied on the L5178 mouse lymphoma cell line using rhodamine 123 accumulation and the MTT cell viability assay. Segetane and jatrophane diterpenes had no cytotoxic activity on the sensitive parent and multidrug resistance cells, while ingenane diterpenes showed a cytotoxic effect on both cell lines. Ingenanes 6: and 7: and segetane 1: demonstrated the remarkable multidrug resistance modulating effect at 20 µM.

Isolation of Phorbol Esters from Euphorbia grandicornis and Evaluation of Protein Kinase C- and Human Platelet-Activating Effects of Euphorbiaceae Diterpenes.

Human platelets contain conventional (α and ß) and novel isoforms of PKC (δ and θ), and PKC activation can result in platelet aggregation and secretion reaction that are important for thrombus formation. Several tumor-promoting Euphorbiaceae diterpenes are known to act as direct activators of PKC, but many types of such diterpenes have not been studied as platelet stimulators. In the present study, two new and five known phorbol esters were isolated from Euphorbia grandicornis. Two of the isolated phorbol esters together with compounds representing ingenane, jatrophane, and myrsinane structural types were studied on PKC activation and platelet stimulation. The investigated phorbol esters and ingenane esters induced blood platelet aggregation and ATP secretion. PKC activation was demonstrated by inducing membrane translocation of PKCs, phosphorylation of PKC substrates, and activation of PKC signaling pathways. The PKC-activating effect of the compounds correlated well with their efficacy to cause platelet stimulation. Moreover, by using an isoform-specific PKC inhibitor, it was found that besides conventional PKCs novel PKCs also play a positive role in platelet activation caused by phorbol/ingenane esters, especially in regulating platelet aggregation. The present results suggest that platelets afford a useful model for studying PKC activators of natural origin or their chemical derivatives.

Diterpene Constituents of Euphorbia exigua L. and Multidrug Resistance Reversing Activity of the Isolated Diterpenes.

Phytochemical investigation of the MeOH extract obtained from the aerial parts of the annual weed Euphorbia exigua L. resulted in the isolation of two novel (1, 2) and one known (3) jatrophane diterpenes. Their structures were established by extensive 1D- and 2D-NMR spectroscopy and HR-ESI-MS. The isolated compounds were evaluated for multidrug resistance (MDR) reversing activity on human MDR gene-transfected L5178 mouse lymphoma cells; and all three compounds were found to modulate the intracellular drug accumulation.

First phytochemical investigation of secondary metabolites of Euphorbia davidii Subils. and antiproliferative activity of its extracts.

The present work is the first phytochemical investigation of Euphorbia davidii Subils. After multistep separation process, three flavonoid glycosides were obtained from the ethyl acetate soluble fraction of the methanol extract of the whole plant. The structures of the isolated compounds were determined as kaempferol 3-O-rhamnoside, myricetin 3-O-rhamnoside, and quercetin 3-O-rhamnoside. Aqueous and organic extracts of the plant were screened in vitro for antiproliferative activity against HeLa (cervix epithelial adenocarcinoma), A431 (skin epidermoid carcinoma), A2780 (ovarian carcinoma) and MCF7 (breast epithelial adenocarcinoma) cells, using the MTT assay. n-Hexane and chloroform extracts demonstrated moderately dose-dependent cell growth inhibitory activity against all four cell lines.

Anticancer Meroterpenoids from Centrapalus pauciflorus leaves: Chromone- and 2,4-Chromadione-Monoterpene Derivatives.

Eight previously undescribed chromones, named pauciflorins F-M and two 5-methyl-2,4-chromadione derivatives named as pauciflorins N and O, were isolated from the methanol extract of the leaves of Centrapalus pauciflorus (Willd.) H.Rob. together with the known (+)-spiro-ethuliacoumarin. The structures were determined via extensive spectroscopic analyses, including HRESIMS, 1D NMR (1H, 13C JMOD), and 2D NMR (HSQC, HMBC, 1H-1H COSY, and NOESY) experiments. Through an MTT assay, seven isolated compounds were tested for their antiproliferative properties against human adherent breast (MCF-7, MDA-MB-231), cervical (HeLa, SiHa), and ovarian (A2780) cancer cell lines. Pauciflorin F was effective against MCF-7 breast cancer cells, its activity (IC50 5.78 µM) was comparable to that of the reference agent cisplatin (IC50 5.78 µM).

Ingol, ent-atisane, and stachane-type diterpenoids from Euphorbia deightonii with multidrug resistance reversing activity.

Nine previously undescribed ingol-type diterpenoid polyesters with eighteen known ingol esters, two ent-atisane, and one stachane diterpenoid were isolated from the methanol extract of Euphorbia deightonii Croizat. The structures were established by extensive spectroscopic analysis involving 1D (1H, 13C J-modulation) and 2D NMR experiments, HRESIMS measurements, and the comparison of the spectroscopic data with reported literature values. The cytotoxic concentrations of 13 isolated compounds were determined, and the compounds were investigated for multidrug resistance modulating activity on an L5178 mouse lymphoma cell line using a rhodamin 123 accumulation assay. Six ingol esters demonstrated the significant inhibition of P-glycoprotein, while the two ent-atisane diterpenoids were found to be inactive. The measured activities allowed to establish some structure-activity relationships. Notably, lower and higher-type diterpenoids simultaneously occurred in E. deightonii.

Triterpenes and Phenolic Compounds from Euphorbia deightonii with Antiviral Activity against Herpes Simplex Virus Type-2.

Two undescribed compounds, 3ß,7ß-dihydroxy-24-methylenelanosta-8-ene-11-one (1) and neolignane deightonin (4) were isolated from the aerial parts of Euphorbia deightonii Croizat together with six known compounds, namely, kansenone (2), euphorbol-7-one (3), dehydrodiconiferyl diacetate (5), marylaurencinol D (6), scoparon (7), and 3,4,3'-tri-O-methylellagic acid (8). The structures of the isolated compounds were determined by HRESIMS, 1D (1H, 13C JMOD) and 2D NMR (HSQC, HMBC, 1H-1H COSY, NOESY) spectroscopic analysis, and by comparison of the assignments with literature data. The anti-herpes simplex virus type-2 activity of the isolated compounds were investigated by qRT-PCR assay on Vero cells after determining cytotoxic concentration 50% (CC50). Compounds 1, 3, 4, and 7 exhibited inhibitory effects with respective IC50 values of 7.05, 2.42, 11.73, and 0.032 µM. Scoparon (7) showed the strongest anti-HSV activity with a selectivity index of 10.93.

Unusual tigliane diterpenes from Euphorbia grandicornis.

Phytochemical study of the aerial parts of Euphorbia grandicornis led to the isolation of two new tigliane diterpenes, 16-angeloyloxy-13α-isobutanoyloxy-4ß,9α,20-trihydroxytiglia-1,5-diene-3,7-dione (1) and 16-angeloyloxy-13α-isobutanoyloxy-4ß,9α,7ß-trihydroxytiglia-1,5-dien-3-one (2). The structures and relative configuration of the new compounds were elucidated on the basis of extensive spectroscopic analysis, including 1D and 2D NMR experiments ((1)H NMR, JMOD, (1)H-(1)H COSY, NOESY, HSQC, and HMBC), mass spectrometry, and comparison with literature data. The biogenesis of 1 and 2 with respect to the unusual 5-en-7-one and 5-en-7-ol moieties is also discussed.

Jatrophane diterpenes from Euphorbia esula as antiproliferative agents and potent chemosensitizers to overcome multidrug resistance.

Phytochemical study of whole, undried plants of Euphorbia esula led to the isolation of six new (1-6) jatrophane diterpene polyesters, named esulatins H-M, together with the known compounds 2α,3ß,5α,7ß,15ß-pentaacetoxy-9α-nicotinoyloxyjatropha-6(17),11-dien-14-one (7), salicinolide (8), and euphosalicin (9). The structures and relative configuration of 1-6 were established on the basis of extensive spectroscopic analysis, including HRESIMS and one- and two-dimensional NMR techniques. All these compounds, together with diterpenes (10-14) isolated previously from this plant, were evaluated for their antiproliferative activity against HeLa, Ishikawa, and MCF7 cells. The multidrug-resistance-reversing activities were also investigated on L5178 mouse lymphoma cells transfected with the pHa MDR1/A retrovirus DNA. Preliminary structure-activity relationship data are discussed.

Bioactive Compounds from Euphorbia usambarica Pax. with HIV-1 Latency Reversal Activity.

Euphorbia usambarica is a traditional medicine used for gynecologic, endocrine, and urogenital illnesses in East Africa; however, its constituents and bioactivities have not been investigated. A variety of compounds isolated from Euphorbia species have been shown to have activity against latent HIV-1, the major source of HIV-1 persistence despite antiretroviral therapy. We performed bioactivity-guided isolation to identify 15 new diterpenoids (1-9, 14-17, 19, and 20) along with 16 known compounds from E. usambarica with HIV-1 latency reversal activity. Euphordraculoate C (1) exhibits a rare 6/6/3-fused ring system with a 2-methyl-2-cyclopentenone moiety. Usambariphanes A (2) and B (3) display an unusual lactone ring constructed between C-17 and C-2 in the jatrophane structure. 4ß-Crotignoid K (14) revealed a 250-fold improvement in latency reversal activity compared to crotignoid K (13), identifying that configuration at the C-4 of tigliane diterpenoids is critical to HIV-1 latency reversal activity. The primary mechanism of the active diterpenoids 12-14 and 21 for the HIV-1 latency reversal activity was activation of PKC, while lignans 26 and 27 that did not increase CD69 expression, suggesting a non-PKC mechanism. Accordingly, natural constituents from E. usambarica have the potential to contribute to the development of HIV-1 eradication strategies.

Inhibition of human cytomegalovirus IE gene expression by dihydro-beta-agarofuran sesquiterpenes isolated from Euonymus species.

The development of strategies intended to inhibit human cytomegalovirus (HCMV) immediate-early (IE) antigen expression is an important goal in research designed to prevent and treat certain forms of cancer. The aim of this study was to identify potent IE antigen-targeting natural compounds as antitumor promoters in an in vitro model of tumor promotion. Nineteen dihydro-beta-agarofuran sesquiterpenes isolated from Euonymus species were evaluated for their ability to inhibit HCMV IE antigen expression in human lung adenocarcinoma (A549) cells. Five esters of penta- and hexahydroxydihydro-beta-agarofuran proved to be active components in these Euonymus species, inhibiting the IE antigen expression of HCMV. The highest activity was displayed by 2beta,6alpha,15-triacetoxy1beta-benzoyloxy-9alpha-nicotinoyloxydihydro-beta-agarofuran. These effective compounds may be regarded as prototypes of antitumor promoters, as secondary chemopreventive agents which can modify or halt tumor promotion in general.

Chemoprevention and inhibition of P-glycoprotein in cancer cells by Chinese medicinal herbs.

Many of the herbal extracts used in the Chinese clinical medical routine inhibit the growth of tumor cells. In the present work, extracts of 12 selected herbs were prepared with methanol, chloroform, ethyl acetate and water, and the effects of these on the multidrug resistance (MDR) and P-glycoprotein of mouse lymphoma cells transfected with the human mdr1 gene and on a human lung alveolar epithelial cell line were investigated. The extracts were tested for antiproliferative effects, and the reversal of MDR in mouse lymphoma cells. The possible chemopreventive effect of the chloroform extracts was studied on the expression of cytomegalovirus (CMV) immediate-early (IE) antigen in human lung cancer cells (A549). The antimicrobial effects of the extracts were tested on some representative micro-organisms. Certain of the chloroform extracts of the plant materials were the most effective compounds on the reversal of MDR. Two of the chloroform extracts enhanced the antiproliferative effect of doxorubicin on MDR mouse lymphoma cells. The selected extracts did not show any antibacterial effect with the agar diffusion method. Certain chloroform extracts decreased the intermediate IE antigen expression of CMV in A459 cells.

New MDR modulators and apoptosis inducers from Euphorbia species.

Several macrocyclic diterpenes with jatrophane or lathyrane skeletons were isolated from methanol extracts of Hungarian Euphorbia species and evaluated for multidrug resistance (MDR)-reversing activity on a human colon cancer cell line. MDR-reversing activity was tested by using a standard functional assay with Rhodamine 123 as a fluorescent substrate analogue of epirubicin. In the model of combination chemotherapy, the interactions between epirubicin and certain resistance modifiers were studied in vitro. Compound 8 proved to be the most active, exhibiting a synergistic interaction. The capacity of the most effective derivative to induce apoptosis was demonstrated by flow cytometric analysis and by staining with ethidium bromide and acridine orange, using human mdrl gene-transfected mouse lymphoma cells and a human cervical adenocarcinoma cell line. The selected diterpene was able to induce moderate apoptosis in the tested cell lines. The data presented here indicate that naturally occurring Euphorbia diterpenes can be regarded as effective lead compounds for the reversal of MDR.

Pellitorine, an extract of Tetradium daniellii, is an antagonist of the ion channel TRPV1.

BACKGROUND: Transient Receptor Potential Vanilloid 1 (TRPV1) confers noxious heat and inflammatory pain signals in the peripheral nervous system. Clinical trial of resiniferatoxin from Euphorbia species is successfully aimed at TRPV1 in cancer pain management and heading toward new selective painkiller status that further validates this target for drug discovery efforts. Evodia species, used in traditional medicine for hundreds of years, are a recognised source of different TRPV1 agonists, but no antagonist has yet been reported. HYPOTHESIS/PURPOSE: In a search for painkiller leads, we noted for the first time a TRPV1 antagonist activity in the fresh fruits of Tetradium daniellii (Benn.) T.G. Hartley (syn. Evodia hupehensis Dode). METHODS: Through a combination of extraction and purification methods with functional TRPV1-specific Ca2+ uptake assays (bioactivity-guided fractionation/isolation/purification); we isolated a new painkiller candidate that is a distant structural homologue of capsiate exovanilloids and endovanilloids such as anandamide, but a putative competitive inhibitor of the TRPV1. Four additional inactive compounds (N-isobutyl-4,5-epoxy-2E-decadienamide, geranylpsoralen, 8-(7',8'-epoxygeranyloxy)psoralen, and xanthotoxol) were also co-purified with pellitorine. Their structures were established by extensive 1D- and 2D-NMR spectroscopic analysis. RESULTS: 1H- and 13C NMR determination of the chemical structure revealed it to be pellitorine, (2E,4E)-N-(2-methylpropyl)deca-2,4-dienamide, which can compete structurally with algesics released in inflammation. In contrast to previous isolates from Evodia species, pellitorine blocked capsaicin-evoked Ca2+ uptake with an IC50 of 154 µg/ml (0.69 mM/l). N-Isobutyl-4,5-epoxy-2E-decadienamide and geranylpsoralen, 8-(7',8'-epoxygeranyloxy)psoralen, and xanthotoxol did not affect the TRPV1. CONCLUSION: This is the first evidence that pellitorine, an aliphatic alkylamide analogue of capsaicin, can serve as an antagonist of the TRPV1 and may inhibit exovanilloid-induced pain.

Discovery and biological evaluation of a new family of potent modulators of multidrug resistance: reversal of multidrug resistance of mouse lymphoma cells by new natural jatrophane diterpenoids isolated from Euphorbia species.

The effects of 15 jatrophane diterpene polyesters (1-3 and 5-16) isolated from lipophilic extracts of Euphorbia serrulata, E. esula, E. salicifolia, and E. peplus (Euphorbiaceae) on the reversion of multidrug resistance of mouse lymphoma cells were examined. The structures of five new compounds (1-5) were elucidated by spectroscopic methods, including HRFABMS, ESIMS, (1)H-(1)H homonuclear and (1)H-(13)C heteronuclear correlations, long-range correlation spectra, and NOESY experiments. The stereochemistry and absolute configuration of one compound (3) were determined by X-ray crystallography. The structure-activity relationship is discussed.

Possible role of fat tissue in the pharmacokinetics of Dodeca-2E,4E,8Z,10E/Z-tetraenoic acid isobutylamides after oral administration of Echinacea angustifolia extract in rats.

Alkamides are one of the most important constituents of lipophilic extracts of Echinacea angustifolia roots. These compounds play an important role in the versatile pharmacological actions of this plant. The present study aimed to compare the concentrations of isomeric dodeca-2E,4E,8Z,10E/Z-tetraenoic acid isobutylamides (DTAI) in brain and periepididymal fat tissues and blood plasma of rats. Thirty, 60, 240 and 720 min after the oral administration of E. angustifolia root extract, tissue and plasma concentrations were determined by reversed-phase HPLC with ESI-MS/MS detection. The calculated terminal t1/2 of the mixture of DTAI was 8.28 h, which indicates a relatively slow elimination. In the 0.5-4 h period the brain/plasma and fat/plasma concentration ratios were continuously above 3 and 18, respectively, followed by equilibrium at 12 h. Our results indicate substantial accumulation of alkamides in lipid-rich tissues, which presumably contributes to a maintained pharmacological action.