Fernando B. Da Costa

Pimarane-type Diterpenes: Antimicrobial Activity against Oral Pathogens

Seven pimarane type-diterpenes re-isolated from Viguiera arenaria Baker and two semi-synthetic pimarane derivatives were evaluated in vitro against the following main microorganisms responsible for dental caries: Streptococcus salivarius, S. sobrinus, S. mutans, S. mitis, S. sanguinis and Lactobacillus casei. The compounds ent-pimara-8(14),15-dien-19-oic acid (PA); ent-8(14),15-pimaradien-3b-ol; ent-15-pimarene-8b,19-diol; ent-8(14),15-pimaradien-3b-acetoxy and the sodium salt derivative of PA were the most active compounds, displaying MIC values ranging from 2 to 8 μg∙mL-1. Thus, this class of compounds seems promising as a class of new effective anticariogenic agents. Furthermore, our results also allow us to conclude that minor structural differences among these diterpenes significantly influence their antimicrobial activity, bringing new perspectives to the discovery of new natural compounds that could be employed in the development of oral care products.

Constituents of glandular trichomes of Tithonia diversifolia: relationships to herbivory and antifeedant activity.

The herbivory activity of the bordered patch larvae (Chlosyne lacinia, Lepidoptera) on leaves of a Brazilian population of Tithonia diversifolia and the antifeedant potential of its leaf rinse extract were investigated. The caterpillars fed only on the adaxial face, where the density of glandular trichomes is very low, and avoided the abaxial face, which contains high levels of trichomes. Deterrent activity against the larvae was observed in leaf discs treated with leaf rinse extract at concentrations of 1-5% of fresh leaf weight. High-performance liquid chromatography (HPLC) analysis indicated that sesquiterpene lactones are the main constituents of the glandular trichomes. Dichloromethane rinse extracts of the leaves and inflorescences were chemically investigated, and 16 compounds were isolated and identified: 14 sesquiterpene lactones, a flavonoid and a diterpenoid. In this study, five sesquiterpene lactones are described for the first time in the genus, including two lactones, one of which has an unusual seco-guaianolide skeleton. Our findings indicate that the caterpillars avoid the sesquiterpene-lactone-rich glandular trichomes, and provide evidence for the antifeedant activity of the dichloromethane leaf rinse extract. In addition, a study of the seasonal variation of the main constituents from the leaf surface throughout a year demonstrated that a very low qualitative but a very high quantitative variation occurs. The highest level of the main metabolite tagitinin C was observed between September and October and the lowest was from March to June, the later corresponding to the period of highest infestation by the larvae.

Anti-inflammatory, analgesic and anti-oedematous effects of Lafoensia pacari extract and ellagic acid.

Lafoensia pacari St. Hil. (Lythraceae) is used in traditional medicine to treat inflammation. Previously, we demonstrated the anti‐inflammatory effect that the ethanolic extract of L. pacari has in Toxocara canis infection (a model of systemic eosinophilia). In this study, we tested the antiinflammatory activity of the same L. pacari extract in mice injected intraperitoneally with β‐glucan present in fraction 1 (F1) of the Histoplasma capsulatum cell wall (a model of acute eosinophilic inflammation). We also determined the anti‐oedematous, analgesic and anti‐pyretic effects of L. pacari extract in carrageenan‐induced paw oedema, acetic acid writhing and LPS‐induced fever, respectively. L. pacari extract significantly inhibited leucocyte recruitment into the peritoneal cavity induced by β‐glucan. In addition, the L. pacari extract presented significant analgesic, anti‐oedematous and anti‐pyretic effects. Bioassay‐guided fractionation of the L. pacari extract in the F1 model led us to identify ellagic acid. As did the extract, ellagic acid presented anti‐inflammatory, anti‐oedematous and analgesic effects. However, ellagic acid had no anti‐pyretic effect, suggesting that other compounds present in the plant stem are responsible for this effect. Nevertheless, our results demonstrate potential therapeutic effects of L. pacari extract and ellagic acid, providing new prospects for the development of drugs to treat pain, oedema and inflammation.

Chlorogenic acids from Tithonia diversifolia demonstrate better anti-inflammatory effect than indomethacin and its sesquiterpene lactones.

ETHNOPHARMACOLOGICAL RELEVANCE T. diversifolia (Hemsl.) A. Gray (Asteraceae) has been used in the traditional medicine in several countries as anti-inflammatory and against other illnesses. It is important to evaluate the anti-inflammatory activity of extracts from the leaves of this species, including an infusion, to identify the main constituents of the extracts, observe their effects and correlate them with the anti-inflammatory activity. MATERIALS AND METHODS An infusion, a leaf rinse extract (LRE) and a polar extract from the rinsed leaves (PE) were obtained and analysed by HPLC-UV-DAD and infrared spectroscopy. The major compounds of these extracts were quantified. The three obtained extracts were evaluated for their anti-inflammatory activities using the paw oedema and croton oil ear oedema assays in mice. Furthermore, neutrophil migration was measured by evaluating myeloperoxidase activity. RESULTS The PE consists primarily of chlorogenic acids (CAs) and lacks sesquiterpene lactones (STLs). The LRE is rich in STLs and includes a few flavonoids. The infusion is chemically similar to the PE but also contains very low amounts of STLs. The PE and LRE have better mechanisms of action than non-steroidal anti-inflammatory drugs (NSAIDs). Unlike NSAIDs, both the PE and LRE inhibit oedema and neutrophil migration. The pool of CAs from the PE of T. diversifolia has an additional mechanism of action, and its anti-inflammatory effect was greater than what is described in the literature for this class of compounds using the same evaluation models. The similar chemical compositions observed for the infusion and the PE, contrasted with the different activities observed, suggests the presence of antagonist compounds produced during the extraction procedure (infusion); the infusion did not inhibit oedema, however it inhibited neutrophil migration. It suggests that although the great majority of plants present CAs, the category of anti-inflammatory effect of their extracts depends on a suitable pool of compounds and an absence of antagonists, among other factors. CONCLUSIONS CAs from T. diversifolia comprise a good pool of anti-inflammatory compounds with better activity mechanisms than NSAIDs, other active compounds from the leaf extracts (STLs and flavonoids) and CAs from other plant sources. Thus, the PE of T. diversifolia has high potential for the development of new anti-inflammatory phytomedicines. The infusion probably contains antagonists, and therefore it can be useful to treat inflammation processes where neutrophil recruitment is involved and oedema is not.

Ethnobotany, Chemistry, and Biological Activities of the Genus Tithonia (Asteraceae)

The genus Tithonia is an important source of diverse natural products, particularly sesquiterpene lactones, diterpenes, and flavonoids. The collected information in this review attempts to summarize the recent developments in the ethnobotany, biological activities, and secondary metabolite chemistry of this genus. More than 100 structures of natural products from Tithonia are reported in this review. The species that has been most investigated in this genus is T. diversifolia, from which ca. 150 compounds were isolated. Biological studies are described to evaluate the anti‐inflammatory, analgesic, antimalarial, antiviral, antidiabetic, antidiarrhoeal, antimicrobial, antispasmodic, vasorelaxant, cancer‐chemopreventive, cytotoxic, toxicological, bioinsecticide, and repellent activities. A few of these studies have been carried out with isolated compounds from Tithonia species, but the majority has been conducted with different extracts. The relationship between the biological activity and the toxicity of compounds isolated from the plants of this genus as well as T. diversifolia extracts still remains unclear, and mechanisms of action remain to be determined.

Effect of the environment on the secondary metabolic profile of Tithonia diversifolia : a model for environmental metabolomics of plants

Tithonia diversifolia is an invasive weed commonly found in tropical ecosystems. In this work, we investigate the influence of different abiotic environmental factors on the plant’s metabolite profile by multivariate statistical analyses of spectral data deduced by UHPLC-DAD-ESI-HRMS and NMR methods. Different plant part samples of T. diversifolia which included leaves, stems, roots, and inflorescences were collected from two Brazilian states throughout a 24-month period, along with the corresponding monthly environmental data. A metabolomic approach employing concatenated LC-MS and NMR data was utilised for the first time to study the relationships between environment and plant metabolism. A seasonal pattern was observed for the occurrence of metabolites that included sugars, sesquiterpenes lactones and phenolics in the leaf and stem parts, which can be correlated to the amount of rainfall and changes in temperature. The distribution of the metabolites in the inflorescence and root parts were mainly affected by variation of some soil nutrients such as Ca, Mg, P, K and Cu. We highlight the environment-metabolism relationship for T. diversifolia and the combined analytical approach to obtain reliable data that contributed to a holistic understanding of the influence of abiotic environmental factors on the production of metabolites in various plant parts.

Antimicrobial ent-pimarane diterpenes from Viguiera arenaria against Gram-positive bacteria

The dichloromethane crude extract from the roots of Viguiera arenaria (VaDRE) has been employed in an antimicrobial screening against several bacteria responsible for human pathologies. The main diterpenes isolated from this extract, as well as two semi-synthetic pimarane derivatives, were also investigated for the pathogens that were significantly inhibited by the extract (MIC values lower than 100 microg mL(-1)). The VaDRE extract was significantly active only against Gram-positive microorganisms. The compounds ent-pimara-8(14),15-dien-19-oic acid (PA); PA sodium salt; ent-8(14),15-pimaradien-3beta-ol; ent-15-pimarene-8 beta,19-diol; and ent-8(14),15-pimaradien-3beta-acetoxy displayed the highest antibacterial activities (MIC values lower than 10 microg mL(-1) for most pathogens). In conclusion, our results suggest that pimaranes are an important class of natural products for further investigations in the search of new antibacterial agents.

Antimicrobial Activity of Kaurane Diterpenes against Oral Pathogens

Two kaurane diterpenes, ent-kaur-16(17)-en-19-oic acid (KA) and 15-β-isovaleryloxy-entkaur- 16(17)-en-19-oic acid (KA-Ival), isolated from Aspilia foliacea, and the methyl ester derivative of KA (KA-Me) were evaluated against oral pathogens. KA was the most active compound, with MIC values of 10 μg mL-1 against the following microorganisms: Streptococcus sobrinus, Streptococcus mutans, Streptococcus mitis, Streptococcus sanguinis, and Lactobacillus casei. However, KA did not show significant activity against Streptococcus salivarius and Enterococcus faecalis, with MIC values equal to 100 and 200 μg mL-1, respectively. Our results show that KA has potential to be used as a prototype for the discovery of new effective anti-infection agents against microorganisms responsible for caries and periodontal diseases. Moreover, these results allow to conclude that minor structural differences among these diterpenes significantly influence their antimicrobial activity, bringing new perspectives to studies on the structure-activity relationship of this type of metabolites with respect to caries and periodontal diseases.

Antimicrobial Activity of Diterpenes from Viguiera arenaria against Endodontic Bacteria

Six pimarane-type diterpenes isolated from Viguiera arenaria Baker and two semi-synthetic derivatives were evaluated in vitro against a panel of representative microorganisms responsible for dental root canal infections. The microdilution method was used for the determination of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against Porphyromonas gingivalis, Prevotella nigrescens, Prevotella intermedia, Prevotella buccae, Fusobacterium nucleatum, Bacteroides fragilis, Actinomyces naeslundii, Actinomyces viscosus, Peptostreptococcus micros, Enterococcus faecalis and Aggregatibacter actinomycetemcomitans. The compounds ent-pimara-8(14),15-dien-19-oic acid, its sodium salt and ent-8(14),15-pimaradien-3β-ol were the most active, displaying MIC values ranging from 1 to 10 μg mL-1. The results also allow us to conclude that minor structural differences among these diterpenes significantly influence their antimicrobial activity, bringing new perspectives to the discovery of new chemicals for use as a complement to instrumental endodontic procedures.

An unusual dimeric guaianolide with antiprotozoal activity and further sesquiterpene lactones from Eupatorium perfoliatum

The CH(2)Cl(2) extract of aerial parts of Eupatorium perfoliatum L. exhibits antiprotozoal activity under in vitro conditions, especially against Plasmodium falciparum (IC(50)=2.7μg/ml). The search for active compounds yielded seven sesquiterpene lactones: Four structurally similar guaianolides, one dimeric guaianolide, and two germacranolides. The guaianolides differ in the degree of oxidation at C-14, ranging from a hydroxyl group up to a free carboxylic acid. The dimeric guaianolide, structurally closely related to the monomers, displays an unusual type of interguaianolide linkage between C-14 and C-4. Except for the germacranolide euperfolitin, all STLs described here were hitherto unknown. Furthermore, the flavonoid aglycones eupafolin, hispidulin, patuletin, and kaempferol were identified in the extract, which, except for kaempferol, have not been described as constituents of E. perfoliatum before. The dimeric guaianolide was shown to be the most active constituent against Plasmodium falciparum (IC(50) = 2.0μM) and was less cytotoxic against rat skeletal myoblasts (IC(50) = 16.2μM, selectivity index of about 8).