Polymorphic characterization and implications on biopharmaceutics properties of potential anti-inflammatory drug candidate eremantholide C from Lychnophora trichocarpha (Brazilian Arnica).

OBJECTIVES: To perform the polymorphic and physicochemical characterization of the potential anti-inflammatory drug, eremantholide C (EREC), as well as to evaluate the influence of these characteristics on its biopharmaceutics classification. METHODS: Eremantholide C was obtained from chloroformic extract of Lychnophora trichocarpha and crystallized in two distinct solvents: chloroform (EREC 1) and ethyl acetate (EREC 2). To evaluate the polymorphism, EREC samples were submitted to melting point, purity, infrared spectroscopy, differential scanning calorimetry (DSC), X-ray powder diffraction, optical microscopy and scanning electron microscopy analysis. In addition, EREC samples crystallized after intrinsic dissolution study were submitted to DSC and X-ray powder diffraction analysis. KEY FINDINGS: EREC 1 showed fusion at 234.7-241.6 °C, while EREC 2 showed fusion at 238.6-243.7 °C. No polymorphic transitions were observed during the intrinsic dissolution experiment. A single sharp endothermic peak was obtained for the EREC samples. X-ray diffraction showed no crystallographic differences between the EREC samples. EREC 1 and EREC 2 showed birefringence under polarized light and indefinite morphology; however, the shape of the crystals was common to the two samples. CONCLUSIONS: Eremantholide C does not present classical or morphological polymorphism; therefore, there is no influence of crystalline transitions in the solubility and consequently in its biopharmaceutics classification and oral absorption process.

Development and validation of a stability indicating method for quantification of the sesquiterpene lactone eremantholide C from Lychnophora trichocarpha (Brazilian arnica)

ABSTRACT Pharmacological activities as anti-inflammatory, anti-hyperuricemic, anti-gouty arthritis, antitumor and trypanocidal activities of the aerial parts from Lychnophora trichocarpha (Spreng.) Spreng. ex Sch.Bip., Asteraceae (Brazilian arnica) have already been proved. Eremantholide C is a sesquiterpene lactone and one of the active chemical constituents responsible for these activities presented by L. trichocarpha. Therefore, the aim of this work was to develop and validate a stability indicating HPLC method for eremantholide C. Eremantholide C stability was evaluated in L. trichocarpha ethanolic extract and in its isolated form. Analytical conditions employed C18 column, acetonitrile/water in gradient elution, flow of 0.8 ml/min at 30 ºC. To correct for the loss of analyte during sample preparation the use of coumarin as an internal standard was necessary. The developed method provides good separation and resolution of the peaks, allowing quantification of eremantholide C, isolated or directly in the ethanolic extract, in internal standard presence. Validation results showed that this method is linear in the concentration range 2-180 µg/ml, precise, accurate and specific. Stability studies showed that L. trichocarpha ethanolic extract and eremantholide C remain stable for 6 months when stored at room temperature and impermeable glass bottle, therefore they can be used safely and effectively within this period. While at 40 ºC there was stability loss, at 8 ºC a stability increase was observed for the extract and the isolated eremantholide C. Forced degradation studies showed that eremantholide C degraded under acidic and alkaline conditions and was stable for three days under neutral and oxidative conditions, and when exposed to high temperature. Thus, with the development of a stability indicative method and the application of it in eremantholide C stability studies, the results can guide the development of new products that adequately preserve the original features of the biologically active substance with quality, safety and efficacy.

In silico and in vitro prediction of gastrointestinal absorption from potential drug eremantholide C.

OBJECTIVES: Analysis of the biopharmaceutical properties of eremantholide C, sesquiterpene lactone with proven pharmacological activity and low toxicity, is required to evaluate its potential to become a drug. METHODS: Preliminary analysis of the physicochemical characteristics of eremantholide C was performed in silico. Equilibrium solubility was evaluated using the shake-flask method, at 37.0 °C, 100 rpm during 72 h in biorelevant media. The permeability was analysed using parallel artificial membrane permeability assay, at 37.0 °C, 50 rpm for 5 h. The donor compartment was composed of an eremantholide C solution in intestinal fluid simulated without enzymes, while the acceptor compartment consisted of phosphate buffer. KEY FINDINGS: Physicochemical characteristics predicted in silico indicated that eremantholide C has a low solubility and high permeability. In-vitro data of eremantholide C showed low solubility, with values for the dose/solubility ratio (ml): 9448.82, 10 389.61 e 15 000.00 for buffers acetate (pH 4.5), intestinal fluid simulated without enzymes (pH 6.8) and phosphate (pH 7.4), respectively. Also, it showed high permeability, with effective permeability of 30.4 × 10-6 cm/s, a higher result compared with propranolol hydrochloride (9.23 × 10-6 cm/s). CONCLUSIONS: The high permeability combined with its solubility, pharmacological activity and low toxicity demonstrate the importance of eremantholide C as a potential drug candidate.

Campomanesia velutina leaves extracts exert hypouricemic effects through inhibition of xanthine oxidase and ameliorate inflammatory response triggered by MSU crystals

ABSTRACT Gout is a destructive arthritis with a high prevalence worldwide. However, the available therapy is not able to increase life quality in many patients. Campomanesia velutina (Cambess) O. Berg, Myrtaceae, is used in Brazilian folk medicine to treat pain, inflammation and rheumatism. The aim of this study was to evaluate the potential of ethanolic and aqueous extracts from C. velutina leaves to treat hyperuricemia and inflammation in gout arthritis model. Ethanolic extract of leaves and aqueous extract of leaves were in vitro assayed on xanthine oxidase inhibitory effect and in vivo on an experimental model of oxonate-induced hyperuricemia in mice, liver xanthine oxidase inhibition and monosodium urate crystal-induced paw edema model. The extracts at both tested doses (100 and 300 mg/kg) reduced serum urate levels. They were also able to inhibit xanthine oxidase in vitro and in vivo, demonstrating that this might be the mechanism of action underlying the urate-lowering effects. In addition, the extracts showed significant anti-inflammatory activity on monosodium urate crystal-induced paw edema, especially aqueous extract (100 and 300 mg/kg) that reduced edema at all evaluated times. Rutin and myricitrin were identified in ethanolic and in aqueous extracts. In this study, myricitrin was able to reduce serum uric acid levels and inhibit liver xanthine oxidase at the dose of 15 mg/kg. The anti-hyperuricemic activity of rutin has been previously reported. Thus, rutin and myricitrin seem to contribute to the observed effects of ethanolic and aqueous extracts. The results demonstrated the ability of aqueous and ethanolic extracts to lower serum urate levels and to reduce edema induced by monosodium urate crystals. Therefore, they may contribute to the management of gout in the future.

Complete assignments of NMR data and assessment of trypanocidal activity of new eremantholide C derivatives.

Chemical transformations of eremantholide C (1), a sesquiterpene lactone that was isolated from Lychnophora trichocarpha Spreng. led to five new derivatives: 1',2'- epoxyeremantholide C (2), 5-n-propylamine-4,5-dihydro-1',2'-epoxyeremantholide C (3), 5-n-propylammonium-4,5-dihydro-1',2'-epoxyeremantholide C chloride (4), 5-n-propylammonium-4,5-dihydroeremantolide C chloride (5) and 16-O-ethyleremantholide C (6). The structures of all these derivatives were assigned on the basis of IR, MS, 1H and 13C NMR data by 1D and 2D techniques. Eremantholide C and the derivatives 2, 4 and 5 were evaluated against trypomastigotes Y and CL strains of Trypanosoma cruzi. Eremantholide C completely inhibited the growth of both the parasites strains while all derivatives were partially active against the CL strain and inactive against the Y strain.

Anti-inflammatory sesquiterpene lactones from Lychnophora trichocarpha Spreng. (Brazilian Arnica).

The aerial parts of Lychnophora trichocarpha Spreng. (Asteraceae) are used macerated in water or ethanol to treat inflammation, pain, rheumatism, contusions, bruises and insect bites in Brazilian traditional medicine. In this study, anti-inflammatory activity of ethanol extract from aerial parts of L. trichocarpha and its ethyl acetate fraction was investigated. Sesquiterpene lactones, lychnopholide (Lyc) and eremantholide C (EreC), isolated of ethyl acetate fraction, were also assayed for in vitro and in vivo anti-inflammatory activity. Topical treatment with ointments containing ethanol extract, its ethyl acetate fraction and sesquiterpene lactones significantly reduced carrageenan-induced mice paw oedema. In vitro assays demonstrated that Lyc inhibited interferon -γ/lipopolysaccharide -stimulated nitric oxide (NO) production in J774A.1 macrophages and increased production of IL-10 anti-inflammatory cytokine. The reduction of tumor necrosis factor-α (TNF-α) production by EreC was accompanied by an increased production of IL-10 in a concentration-dependent manner in J774A.1 macrophages. The anti-inflammatory effect of Lyc seems to involve the inhibition of production of NO and increased production of IL-10. The mechanism of the effect of EreC on the reduction of carrageenan-induced paw oedema may be attributed to inhibition of production of TNF-α and stimulation of IL-10 production. The results corroborate the use of ethanol extract from Lychnophora trichocarpha in folk medicine for anti-inflammatory action and indicate that the topical route is suitable for use.

Anti-inflammatory and antinociceptive activities of Campomanesia adamantium.

ETHNOPHARMACOLOGICAL RELEVANCE: Campomanesia species are used in folk medicine as anti-inflammatory, anti-rheumatic, anti-diarrheal and hypocholesterolemic. AIM OF THE STUDY: The present study investigated the in vivo anti-inflammatory and antinociceptive properties of ethyl acetate (AE) and aqueous (Aq) extracts from leaves of Campomanesia adamantium and in vitro anti-inflammatory activity of AE and its isolated flavonols, myricitrin and myricetin. MATERIALS AND METHODS: The antinociceptive activity of AE and Aq was evaluated using acetic acid-induced writhing and formalin methods. The in vivo anti-inflammatory effect of AE and Aq was evaluated using carrageenan-induced paw oedema in mice. AE, myricitrin and myricetin were evaluated for their abilities to modulate the production of NO, TNF-α and IL-10 in LPS/IFN-γ stimulated J774.A1 macrophages. RESULTS: It was found that orally administrated AE and Aq (125 and 250 mg/kg) inhibited carrageenan-induced paw oedema in mice. AE (125 and 250 mg/kg) and Aq (125 mg/kg) reduced the time to licking at the second phase of the formalin method in vivo in mice. AE (250 mg/kg) and Aq (125 mg/kg) also reduced the number of writhes. AE, myricitrin and myricetin inhibited NO (320 µg/mL and 6.25-100 µM, respectively) and TNF-α production by macrophages (320 µg/mL for AE, 100 µM for myricitrin and 25-100 µM for myricetin). AE (160 and 320 µg/mL), myricitrin (50 and 100 µM) and myricetin (25-100 µM) increased IL-10 production by macrophages. CONCLUSIONS: The ethyl acetate and aqueous extracts from Campomanesia adamantium showed antinociceptive and anti-inflammatory effects supporting the use of the plant in folk medicine. The results suggest that anti-oedematogenic effect promoted by aqueous extract involves several anti-inflammatory mechanisms of action. The antinociceptive effect shown by aqueous extract can be due to the modulation of release of inflammatory mediators involved in nociception. The anti-inflammatory effects of AE and of its isolated flavonols may be attributed to inhibition of pro-inflammatory cytokines production, TNF-α and NO and to the increased of IL-10 production.

Toxicological evaluation of ethanolic extract of Lychnophora trichocarpha, Brazilian arnica

The species of the genus Lychnophora, Asteraceae, are popularly known as "arnica" and are native from Brazilian savana (Cerrado). They are widely used in Brazilian folk medicine as anti-inflammatory, to treat bruise, pain, rheumatism and for insect bites. For evaluation of acute toxicity, the ethanolic extract was given to albino female and male mice. In open-field test, the extract of Lychnophora trichocarpha (Spreng.) Spreng. (0.750 g/kg) induced a significant inhibition of the spontaneous locomotor activity and exploratory behavior of the animals were observed 1 and 4 h after administration. In traction test, the same dose reduced the muscular force 1 h after administration. The exploratory behavior reduced significantly in the group that received 0.50 g/kg, 1 and 4 h after administration of the extract. The animals that received the doses of 0.25, 0.50 and 0.75 g/kg did not show any change of blood biochemical parameters comparing to control group and showed some histopathological changes such as congestion and inflammation of kidney and liver. The dose of 1.5 g/kg caused the most serious signs of toxicity. Histopathological changes observed was hemorrhage in 62.5% and pulmonary congestion in 100% of the animals. Brain and liver congestion was found in 62.5% of the animals.

Complete assignments of 1H and 13C NMR data for trypanocidal eremantholide C oxide derivatives.

The chemical transformations of eremantholide C (1), a trypanocidal sesquiterpene lactone isolated from Lychnophora trichocarpha Spreng., gave five new oxide derivatives: 3'-hydroxyeremantholide C (2), 1'-formyleremantholide C (3), 1'-carboxyeremantholide C (4), 1'-carbomethoxyeremantholide C (5) and sodium 1'-carboxylate of eremantholide C (6). The (1)H and (13)C NMR data of all these derivatives were assigned based on 1D and 2D techniques. The derivatives were evaluated against Y and CL strains of Trypanosoma cruzi. All of them were inactive against the Y strain. Compounds 2 and 5 displayed 100% activity on the CL strain while compounds 4 and 6 were partially active on the CL strain.

Substâncias da natureza com atividade anti-Trypanosoma cruzi / Natural compounds with anti-Trypanossoma cruzi activity

A doença de Chagas, uma zoonose causada por Trypanosoma cruzi, afeta em todo o mundo cerca de 16 a 18 milhões de pessoas. Sua transmissão ocorre através das fezes de triatomíneos, insetos hematófagos conhecidos como barbeiro. Atualmente, a principal forma de transmissão da doença de Chagas em áreas urbanas é por meio de transfusão de sangue contaminado. A violeta de genciana é o único agente que pode ser empregado na quimioprofilaxia de sangue destinado à transfusão. No entanto, existem algumas restrições ao seu uso. A quimioterapia disponível para a doença de Chagas não é eficaz uma vez que as drogas disponíveis nifurtimox e benznidazol são ativas apenas na fase aguda da doença e apresentam sérios efeitos colaterais. Várias substâncias isoladas de plantas foram avaliadas como agentes anti-T. cruzi, objetivando encontrar drogas com menos efeitos colaterais e maior eficácia para a quimioprofilaxia e quimioterapia da doença de Chagas. Nesta revisão são apresentadas as substâncias de origem natural com atividade anti- T. cruzi.

Chagas'disease, a zoonose caused by Trypanosoma cruzi, affects 16-18 million people in the world. The most important mode of transmission of the disease is associated with the feces of several species of triatomine bugs that are strictly hematophagous. Actually, infected blood transfusion is the major mechanism of transmission in urban areas. Gentian violet is the only available prophylactic drug. Despite its effectiveness, there are some restrictions on its use. The available therapy of Chagas' disease is inadequate since the treatment of patients with the drugs nifurtimox and benznidazole presents serious toxic side effects. The search for new trypanocidal compounds to the treatment of Chagas'disease and to use for eliminating T. cruzi from blood, that are more effective and that do not affect red blood cells is the main goal in the prevention of Chagas' disease. In this review a large set of chemicals of plant origin are enumerated and their trypanocidal activity are briefly discussed.