Pharmacological insight into the anti-inflammatory activity of sesquiterpene lactones from Neurolaena lobata (L.) R.Br. ex Cass.

PURPOSE: Neurolaena lobata is a Caribbean medicinal plant used for the treatment of several conditions including inflammation. Recent data regarding potent anti-inflammatory activity of the plant and isolated sesquiterpene lactones raised our interest in further pharmacological studies. The present work aimed at providing a mechanistic insight into the anti-inflammatory activity of N. lobata and eight isolated sesquiterpene lactones, as well as a structure-activity relationship and in vivo anti-inflammatory data. METHODS: The effect of the extract and its compounds on the generation of pro-inflammatory proteins was assessed in vitro in endothelial and monocytic cells by enzyme-linked immunosorbent assay. Their potential to modulate the expression of inflammatory genes was further studied at the mRNA level. In vivo anti-inflammatory activity of the chemically characterized extract was evaluated using carrageenan-induced paw edema model in rats. RESULTS: The compounds and extract inhibited LPS- and TNF-α-induced upregulation of the pro-inflammatory molecules E-selectin and interleukin-8 in HUVECtert and THP-1 cells. LPS-induced elevation of mRNA encoding for E-selectin and interleukin-8 was also suppressed. Furthermore, the extract inhibited the development of acute inflammation in rats. CONCLUSIONS: Sesquiterpene lactones from N. lobata interfered with the induction of inflammatory cell adhesion molecules and chemokines in cells stimulated with bacterial products and cytokines. Structure-activity analysis revealed the importance of the double bond at C-4-C-5 and C-2-C-3 and the acetyl group at C-9 for the anti-inflammatory activity. The effect was confirmed in vivo, which raises further interest in the therapeutic potential of the compounds for the treatment of inflammatory diseases.

Prenylated flavonoid derivatives from the bark of Erythrina addisoniae.

Two new prenylated flavanones, 2 S-3'-(2-hydroxy-3-methylbut-3-enyl)licoflavone-4'-methyl ether ( 3) and 2 S-3'-(2-hydroxy-3-methylbut-3-enyl)abyssinone II ( 4), and four known flavanones ( 1, 2, 5, 6) were isolated from the stem bark of Erythrina addisoniae. The structures were elucidated on the basis of their spectroscopic and physicochemical data. None of the compounds showed antioxidative properties. 4'-Methylabyssinone V ( 1) and abyssinoflavanone VII ( 6) showed moderate cytotoxic activity (IC 50 = 5 and 3.5 micromol/L, respectively), but apoptosis (caspase-3/7-activation, nuclear fragmentation) was selectively induced by abyssinoflavanone VII ( 6).

Pterocarpans phaseollin and neorautenol isolated from Erythrina addisoniae induce apoptotic cell death accompanied by inhibition of ERK phosphorylation.

The genus Erythrina (Leguminosae), consisting of over 100 different species, is distributed in tropical regions. In traditional medicine, Erythrina species are used to treat cancer, but little is known about the anticancer mechanisms. From the stem bark of Erythrina addisoniae Hutch. & Dalziel, six prenylated pterocarpans were isolated and analysed for pharmacological activity: While calopocarpin, cristacarpin, orientanol c, and isoneorautenol showed only a weak or moderate toxicity in H4IIE hepatoma cells (EC(50)-value> 25 microM), the toxicity of neorautenol and phaseollin was in the low micromolar range (EC(50)-value: 1 and 1.5 microM, respectively). We further focused on these two substances showing that both increased caspase 3/7 activity and nuclear fragmentation as markers for apoptotic cell death. Neorautenol (10 microM, 2h), but not phaseollin induced the formation of DNA strand breaks (comet assay). Both substances showed no effect on NF-kappaB signalling (SEAP assay: basal activity and stimulation with TNF-alpha), on the other hand both pterocarpans (10 microM, 2 h) decreased the activation of the ERK kinase (p44/p42), an mitogen activated protein kinase which is associated with cell proliferation. We conclude that the pterocarpans phaseollin and neorautenol may be responsible for the anticarcinogenic actions of the plant extract reported in the literature. Further analysis of these substances may lead to new pharmacons to be used in cancer therapy.

Antiprotozoal activity of Neurolaena lobata.

Extracts, fractions and sesquiterpene lactones from Neurolaena lobata (L.) R. Br. (Asteraceae), a traditional medicinal plant from Guatemala, were tested in vitro against Leishmania spp. promastigotes, Trypanosoma cruzi trypomastigotes and epimastigotes and Trichomonas vaginalis trophozoites. The ethanol extract inhibited the parasite growth of L. mexicana, T. cruzi and T. vaginalis significantly. The pure germacranolides 1 and a mixture of 2 and 3, isolated from the ethonal extract, were highly active against L. mexicana and T. cruzi.

Insecticidal activity of essential oils to the tobacco cutworm, Spodoptera litura.

Twenty-one essential oils were tested for insecticidal activity via topical administration to third instar larvae of the tobacco cutworm, Spodoptera litura. Oils of Satureia hortensis, Thymus serpyllum and Origanum creticum produced >90% larval mortality at 24 h at a dose of 100 microg per larva. The LD(50) value for S. hortensis (48.4 microg) was comparable to that for Thymus vulgaris (46.9 microg). The monoterpenoid phenols thymol and carvacrol, major constituents of the oils of Thymus and Satureia species, likely account for the insecticidal action of the species.

Furanoheliangolides from leaves of Neurolaena macrocephala.

Six sesquiterpene lactones, two new (3 and 6) and four known (1, 2, 4 and 5) were isolated from the leaves of the Mexican Neurolaena macrocephala (Asteraceae). The furanoheliangolide 6, containing 4beta,5-dihydro-9alpha-hydroxy-atripliciolide as basic structure, was found for the first time in nature. The chemotaxonomic importance of this phytochemical work is discussed.

Sesquiterpene lactone glycosides from Arnica longifolia.

Flowers of ARNICA LONGIFOLIA were investigated for the occurrence of sesquiterpene lactone glycosides. A mixture containing the two isomeric 11alpha,13-dihydro- and 11beta,13-dihydro-4 H-tomentosin glucosides, previously isolated from A. AMPLEXICAULIS and A. MOLLIS, together with the two isomeric 11alpha,13-dihydro- and 11beta,13-dihydro-4 H-carabrone glucosides were found in A. LONGIFOLIA. All carbon and proton shifts of the glycosides could be assigned by their 2D-COSY and 2D-HETCOR spectra and spectroscopy of their respective aglycones, obtained via enzymatic hydrolysis.

Plants used in Guatemala for the treatment of protozoal infections: II. Activity of extracts and fractions of five Guatemalan plants against Trypanosoma cruzi.

The activities of crude plant extracts of five plants popularly used in Guatemala against bacterial and protozoal infections and some of their fractions have been evaluated against the trypomastigote and epimastigote forms of Trypanosoma cruzi in vitro. The most active fraction of Neurolaena lobata has also been screened in vivo. Main in vitro activities against trypomastigotes have been observed for the hexane and ethanol extracts of N. lobata (Asteraceae). Both extracts were also active against epimastigotes, whereas all other extracts tested had no effect on epimastigotes. For the hexane extracts of Petiveria alliacea (Phytolaccaceae) and Tridax procumbens (Asteraceae) a marked inhibition of trypomastigotes has been found. Also the ethanol extracts of Byrsonima crassifolia (Malpighiaceae) leafs and Gliricidia sepium (Papilionaceae) bark showed some trypanocidal activity. Fraction 2 of the ethanol extract of N. lobata was highly active against T. cruzi as well in vitro as in vivo. The chloroforme fraction of P. alliacea showed a high inhibition of trypomastigotes in vitro. Also three fractions of the active extract of B. crassifolia inhibited T. cruzi trypomastigotes. No fraction of G. sepium bark extract showed a marked trypanocidal activity.

Antimicrobial properties of Honduran medicinal plants.

Ninety-two plants used in the traditional pharmacopoeia of the Pech and neighboring Mestizo peoples of central Honduras are reported. The results of in vitro antimicrobial screens showed that 19 of the extracts from medicinal plants revealed signs of antifungal activity while 22 demonstrated a measurable inhibitory effect on one or more bacterial cultures. Bioassay-guided fractionation of extracts from Mikania micrantha, Neurolaena lobata and Piper aduncum produced weak to moderately active isolates. The broad spectrum of activity of the extracts helps to explain the widespread use of these plants for wound healing and other applications.

Variability of sesquiterpene lactones in Neurolaena lobata of different origin.

Leaves of Neurolaena lobata (L.) R. Br. originating from Guatemala, were analyzed using HPLC for their qualitative and quantitative sesquiterpene lactone contents. Significant differences in the individual amounts of neurolenins and furanoheliangolides were found between four natural populations. When plants were cultivated on proving fields at two different localities in Guatemala, their sesquiterpene lactone patterns matched the natural population, but differed quantitatively. The meaning of these differences for the use of N. lobata in traditional medicine and its cultivation is discussed.

Antiplasmodial activities and cytotoxic effects of aqueous extracts and sesquiterpene lactones from Neurolaena lobata.

Aqueous and lipophilic extracts of Neurolaena lobata (Asteraceae), obtained from Guatemala, were tested against Plasmodium falciparum in vitro. Moreover, sesquiterpene lactones, of the germacranolide and furanoheliangolide type, isolated from N. lobata, were shown to be active against P. falciparum in vitro. In addition to their antiplasmodial activity, their cytotoxic effects on human carcinoma cell lines were evaluated. Structure-activity relationships are discussed.

11Alpha,13-and 11beta,13-dihydro-4H-xanthalongin 4-O-beta-glucopyranosides: new sesquiterpene lactone glycosides from flowers of Arnica amplexicaulis and A. mollis.

The flowers of A. amplexicaulis afforded, in addition to xanthalongin ( 1), 11beta,13-dihydroxanthalongin (2), its 11-epimer (3), and 11alpha,13-dihydro-4H-xanthalongin (4), the new xanthanolide glycosides 11alpha, 13-dihydro-4 H-xanthalongin 4- O-beta-D-glucopyranoside (5) and its 11-epimer (6). The glycosides were also isolated from the flowers of A. mollis. The structures were elucidated by NMR spectroscopy (1)H-NMR, COSY, (13)C-NMR, HETCOR, DEPT), MS (EI, FAB, FD), and enzymatic hydrolysis of the glycosides. The chemo-taxonomic importance is discussed.

Decreased helenalin-induced cytotoxicity by flavonoids from Arnica as studied in a human lung carcinoma cell line.

The effect of the flavones apigenin, luteolin, hispidulin and eupafolin, and of the flavonols kaempferol, quercetin, 6-methoxykaempferol and patuletin from Arnica spp. on the cytotoxicity of the sesquiterpene lactone helenalin was studied in the human lung carcinoma cell line GLC4 using the microculture tetrazolium (MTT) assay. The tumour cells were exposed to the test compounds for 2h. Helenalin concentrations around its control IC(50) value, 0.5 µM, were combined with flavonoid concentrations ranging from 0.01 to 20 µM. At non-toxic concentrations, up to 10µM, all flavonoids except kaempferol significantly reduced the helenalin-induced cytotoxicity. Hispidulin and patuletin displayed their modulating effect on helenalin-induced cytotoxicity in the broadest concentration range. The strongest effect was found with 5 and 10µM hispidulin, 0.05 µM quercetin, and 1 µM patuletin, increasing the IC(50) value of helenalin with circa 40%. No dose-dependency was found in the concentration range tested.

Cytotoxicity of flavonoids and sesquiterpene lactones from Arnica species against the GLC4 and the COLO 320 cell lines.

The cytotoxicity of 21 flavonoids and 5 sesquiterpene lactones, as present in Arnica species, was studied in GLC4, a human small cell lung carcinoma cell line, and in COLO 320, a human colorectal cancer cell line, using the microculture tetrazolium (MTT) assay. Following continuous incubation, most flavonoids showed moderate to low cytotoxicity, as compared with the reference compound cisplatin (IC50 = 1.1 microM against GLC4 and 2.9 microM against COLO 320). Their IC50 values varied from 17 to > 200 microM. The most toxic compound was the flavone jaceosidin. Of the sesquiterpene lactones tested, helenalin, possessing both the reactive alpha-methylene-gamma-lactone moiety and a reactive alpha,beta-unsubstituted cyclopentenone ring, displayed the strongest cytotoxicity. For 2 h exposure, its IC50 value was 0.44 microM against GLC4 and 1.0 microM against COLO 320. COLO 320 was more sensitive than GLC4 for many flavonoids (especially for flavones), but more resistant to the cytotoxic effect of the sesquiterpene lactones bearing an exocylic methylene group fused to the lactone function.

Tussilagine and isotussilagine: two pyrrolizidine alkaloids in the genus arnica1.

In flowerheads of ARNICA MONTANA, A. CHAMISSONIS ssp. FOLIOSA, A. AMPLEXICAULIS, and A. SACHALINENSIS, traces of the non-toxic pyrrolizidine alkaloids tussilagine and isotussilagine were detected. Their identity was unambiguously confirmed by direct comparison (TLC, GC, GC/MS) with the authentic samples.

[Allergic contact dermatitis caused by Asteraceae. Identification of an 8,9-epoxythymol-diester as the contact allergen of Arnica sachalinensis]. / Allergische Kontaktdermatitis auf Asteraceae. Identifizierung eines 8,9-Epoxythymoldiesters als Kontaktallergen von Arnica sachalinensis.

A pharmacist developed an allergic contact dermatitis while working with Arnica sachalinensis. Patch testing with sesquiterpene lactones was negative. The contact allergen of A. sachalinensis was identified as 10-acetoxy-8,9-epoxy-thymol-isobutyrate. This compound, or structurally related 8,9-epoxythymol-diesters, are often found in Asteraceae. In addition to sesquiterpene lactones, epoxythymol-diesters should be considered as contact allergens. Cross reactions between the two groups of compounds are not expected and could not be demonstrated in our patients.