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5'-methoxynobiletin (5'-MeONB), a polymethoxyflavone isolated from A. conyzoides, has shown anti-inflammatory property. Nevertheless, the antinociceptive activity and pre-clinical pharmacokinetics (PK) characteristics of 5'-MeONB remain unknown. Considering the anti-inflammatory potential of the 5'-MeONB, this study aimed to investigate the pre-clinical PK behavior of 5'-MeONB, as well as its time course antinociceptive activity.5'-MeONB plasma concentrations were determined in Wistar rats after intravenous (i.v.) (10 mg/kg) and oral (50 mg/kg) administration, and in Swiss mice after oral administration (100 mg/kg). Plasma samples were deproteinization and 5'-MeONB quantified by a validated UPLC-MS method. Additionally, the antinociceptive activity of 5'-MeONB was evaluated after 15, 30, 60, 180 and 360 min following oral administration on the acute nocifensive behavior of mice induced by formalin.5'-MeONB rats and mice plasma concentration-time profiles were best one-compartment model. After i.v. administration to rats, a short half-life, a high clearance and moderate volume of distribution at steady state were observed. Similar results were obtained after oral administration. The oral bioavailability ranged from 8 to 11%. Additionally, 5'-MeONB exhibited antinociceptive activity in both formalin phases, especially in the inflammatory phase of the model, inhibiting 68% and 91% of neurogenic and inflammatory responses, respectively, after 30 min of oral administration.The results described here provide novel insights on 5'-MeONB pharmacokinetics and pharmacodynamic effect, serving as support for future studies to confirm this compound as anti-nociceptive and anti-inflammatory effective agent.

Ethnopharmacological relevance Myrocarpus frondosus, known as cabreuva, is a tree whose trunk barks are used in folk medicine as tea, syrup, ointments, and tinctures for the treatment of inflammation. However, there is no scientific evidence demonstrating this activity. Aim of the study The present investigation was focused on evaluating the antioxidant and anti-inflammatory activities of M. frondosus, using the in vitro model of RAW 264.7 macrophages induced by LPS and the in vivo model of mouse pleurisy induced by carrageenan. Materials and methods M. frondosus trunk barks were dried at room temperature for seven days and subjected to exhaustive maceration with ethanol (70%) to obtain its crude extract (CE). CE was subjected to UPLC-HRMS analysis to establish its chemical profile. Its antioxidant activity was evaluated using the DPPH method, reducing power by the iron (III) to iron (II) reduction assay and the β-carotene-linoleic acid bleaching assay. The RAW 264.7 macrophages were pretreated with the CE in a non-cytotoxic concentration and induced by LPS (1 μg/mL). After 24 h, using the supernatant, we evaluated the nitric oxide (NOx) and interleukin-6 (IL-6) levels. The anti-inflammatory effects of CE (at doses of 30, 100 and 300 mg/kg) were evaluated on leukocyte migration (total and differential), exudate concentrations, myeloperoxidase (MPO) and adenosine-deaminase (ADA) activities, NOx, tumor necrosis factor-α (TNF-α), and IL-6 levels, by using a murine model of neutrophilic inflammation. Results The UPLC-HRMS of CE revealed the presence of isoflavonones, including biochanin A and formononetin. CE exhibited good antioxidant activity by quenching and decreasing free radicals, as well as reducing pro-oxidant metals. CE did not show cytotoxicity at a concentration below 11 μg/mL and reduced the secretion of the pro-inflammatory NOx in the inflamed macrophages. In vivo assay revealed that CE caused a pronounced inhibition on leukocyte migration, and this inhibition was due to its ability to reduce neutrophil migration. Moreover, CE was also able to reduce the release of critical pro-inflammatory mediators such as MPO, NOx, TNF-α, and IL-6. Conclusions All these findings indicate that M. frondosus exhibited antioxidant activity and anti-inflammatory effect.

The present study characterized propolis extracts produced by Scaptotrigona bipunctata (Tubuna) and Melipona quadrifasciata (Mandacaia) by LC–MS/MS; their cytotoxicity as well as the mechanism of action in a melanoma cellular model were also assessed. The chemical characterization performed by UPLC-ESI-QTOF/MS2 analysis revealed uncommon presence of piperidinic alkaloids in Tubuna’s propolis extract together with C-glycopyranoside flavonoids. Mandacaia’s propolis collected in the same area rather presented terpenoids and flavonoids. Regarding the mechanism of cytotoxicity, propolis extracts increased the accumulation of reactive oxygen species (ROS), reduced the potential of mitochondrial membrane, induced a decrease in the proteins Bcl-2 and AKT-3 levels, and decreased melanoma cells’ migration and invasion. Both propolis extracts induced apoptosis while only Mandacaia’s propolis extract induced cell cycle arrest in G2/M.

Ageratum conyzoides L. is a plant widely used in traditional medicine of tropical and subtropical regions for its anti-inflammatory and antinociceptive properties. Nevertheless, the chemical composition of its medicinal preparation has not been yet accurately established. In this study, chromatographic methods were developed for the simultaneous identification and quantification of the main compounds in A. conyzoides aqueous extract, using UPLC-PDA-ESI-QToF-MS. The qualitative analyses defined by MS/MS analysis allowed the identification of 27 compounds in the aqueous extract, including the toxic pyrrolizidine alkaloids, phenolic acids, coumarin and polymethoxyflavones. Among the metabolites, twelve were detected for the first time in the species. The quantitative method was validated according to the official guidelines, demonstrating to be specific, linear, precise, accurate and sensitive for the quantification of chlorogenic acid, coumaric acid, coumarin (1,2-benzopyranone), 5,6,7,3',4',5'-hexamethoxyflavone, nobiletin, 5'-methoxynobiletin and eupalestin.

Jambu [Acmella oleracea (L.) R.K. Jansen] is an edible plant with a wide range of constituents of biological interest. In this study, the chemical composition of leaves, flowers and stems of jambu cultivated in hydroponic and conventional systems was investigated. In both crop systems, the leaves showed the highest total phenolic content, total flavonoid content and in vitro antioxidant capacity. The extracts were characterized by determining 45 compounds, including phenolic acids, glycosylated flavonoids, alkamides and fatty acids, by LC-MS analysis. Of these compounds, 31 are described for the first time in this species, five of which are reported for the first time in the literature. The PCA and cluster analysis results distinguished different anatomical parts (PC1 and PC2) and cultivation systems (PC3) into well-defined groups.

Ageratum conyzoides Linn (Asteraceae), a tropical plant that is very common in West Africa and some parts of Asia and South America, has been used to treat inflammatory disorders. In Brazil, teas made from A. conyzoides L. are used as anti-inflammatory, analgesic and anti-diarrheic agents. Therefore, it is necessary to study the mechanism of anti-inflammatory action of A. conyzoides L. to support its medicinal use for treating inflammatory conditions. These studies will also support the development of effective pharmacological agents with potent anti-inflammatory properties.To evaluate the anti-inflammatory effects of the crude extract (CE), its derived fractions: ethanol (EtOH-F), hexane (HEX-F), ethyl acetate (EtOAc-F) and dichloromethane (DCM-F) and isolated compounds, such as 5'-methoxy nobiletin (MeONOB), 1,2-benzopyrone and eupalestin, which are obtained from the aerial parts of A. conyzoides L.These evaluations were performed using an animal model of inflammation induced by carrageenan. The following inflammatory parameters were analysed: leukocyte influx, protein concentration of the exudate, myeloperoxidase (MPO), adenosine deaminase (ADA) and nitric oxide metabolites (NOx) concentrations, interleukin 10 (IL-10), interleukin 17A (IL-17A), interleukin 6 (IL-6), tumor necrosis factor (TNF), interferon gamma (IFN-γ) and phosphorylation of p65 subunit of NF-κB (p-p65 NF-κB), p38 mitogen-activated protein kinases (p-p38 MAPK) were also analysed.CE, its EtOH-F, HEX-F, EtOAc-F and DCM-F and the isolated compounds, including MeONOB, 1,2-benzopyrone and eupalestin, significantly reduced leukocyte influx, protein concentration of the exudate, MPO, ADA, and NOx concentrations (p0.05). CE, EtOH-F and isolated compounds significantly reduced IL-17A, IL-6, TNF and IFN-γ levels (p0.05). CE, EtOH-F and isolated compound 1,2-benzopyrone also increased IL-10 levels (p0.05). Isolated compounds, MeONOB, 1,2-benzopyrone and eupalestin, reduced p-p65 NF-κB and p-p38 MAPK (p0.01).This study demonstrates that A. conyzoides L. exerts its important anti-inflammatory properties by inhibiting leukocyte influx and protein concentration of the exudate, as well as reducing the levels of several pro-inflammatory mediators. The anti-inflammatory action of A. conyzoides L. may be because of the inhibition of p65 NF-κB and MAPK activation by the isolated compounds.

Jungia sellowii (Asteraceae) is a shrub that grows in Southern Brazil and polar extract of its leaves presents anti-inflammatory properties. Cyperane, guaiane, nortrixane, and trixane sesquiterpene types were reported as the main metabolites in Jungia species. This work aims to describe the isolation and identification of sesquiterpenes in the leaves of J. sellowii using liquid–liquid partition and centrifugal partition chromatography. Thus, the crude extract of fresh leaves of J. sellowii was partitioned with hexane, dichloromethane, ethyl acetate and butanol, respectively. The butanol fraction was then subjected to a selected ternary system optimized for the CPC (centrifugal partition chromatography): ethyl acetate–ethanol–water (9:2:10, v/v/v). The separation was carried out isocratically at a flow rate of 25 mL/min at 1200 rpm, affording seven fractions A to G. TLC of fractions B, C and F displayed a single spot corresponding to three new glycosylated sesquiterpenoids. Their structures were established by using spectroscopic data in comparison to those reported in the literature. Furthermore, the isolates were evaluated for their leishmanicidal and cytotoxic effects. No cytotoxic effect was observed against the three cancer cell lines (HL60, JURKAT and REH), but compound 1 showed a weak antiprotozoal activity. Liquid–liquid partition and CPC turned to be a versatile technique of glycoside purification which is environmentally friendly and requires a limited amount of organic solvents.

This study presents the development and validation of a fast and simple bioanalytical ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS) method intended for quantifying the anti-inflammatory candidate 5′-methoxynobiletin (5′-MeONB) in rat plasma. Standard of 5′-MeONB was purified from A. conyzoides extract by using preparative HPLC. After a pretreatment of plasma samples with acetonitrile, chromatographic separations were efficiently achieved with a C18 column using a 9 min gradient system of 0.1% aqueous formic acid and acetonitrile as eluent. Drug candidate 5′-MeONB and chrysin (internal standard, IS) detection were carried out using ESI+ through the extracted ion chromatograms approach, monitored at m/z 433.1494 (for 5′-MeONB, tR:1.78 min) and m/z 255.0657 (for IS, tR:1.57 min). Method was validated according to US FDA guidelines, presenting linearity (R2 > 0.999) over concentration range of 30–750 ng/mL. Relative standard deviation (RSD) of repeatability and intermediary precision respectively ranged between 1.93–3.65% and 2.16–7.54%, considering lower limit of quantitation (30 ng/mL) and quality control (90, 360 and 600 ng/mL) samples, while accuracy was between 82.51 and 109.44%. Moreover, no interference from plasma endogenous substances, no carryover effect, and no influence of extraction method even in hemolyzed blood samples were observed. Sample stability in auto-sampler and long-term −80 °C storage, as well as matrix effect were within acceptable limits. For the first time, using the validated UPLC-MS bioanalytical method, the plasma pharmacokinetics of 5′-MeONB following 2 mg/kg intravenous bolus dosing to Wistar rats was characterized allowing the determination of the parameters describing drug distribution and elimination.

Vernonanthura tweedieana (Baker) H. Rob., Asteraceae, is used in the Brazilian folk medicine for the treatment of respiratory diseases. In this work the phytochemical investigation of its ethanol extracts as well as the development and validation of an UPLC-PDA method for the quantification of the eriodictyol from the leaves were performed. The phytochemical study for this species lead to the identification of ethyl caffeate, naringenin and chrysoeriol in mixture, eriodictyol from leaves, and the mixture of 3-hydroxy-1-(4-hydroxy-3,5-dimethoxyphenyl)-propan-1-one and evofolin B, apigenin, the mixture of caffeic and protocatechuic acid and luteolin from stems with roots, being reported for the first time for V. tweedieana, except for eriodictyol. The structural elucidation of all isolated compounds was achieved by 1H and 2D NMR spectroscopy, and in comparison with published data. An UPLC-PDA method for quantification of the eriodictyol in leaves of V. tweedieana was developed and validated for specificity, linearity, precision (repeatability and intermediate precision), limit of detection (LOD) and limit of quantification (LOQ), accuracy and robustness. In this study, an excellent linearity was obtained (r2 = 0.9999), good precision (repeatability RSD = 2% and intermediate precision RSD = 8%) and accuracy (average recovery from 98.6% to 99.7%). The content of eriodictyol in the extract of leaves of V. tweedieana was 41.40 ± 0.13 mg/g. Thus, this study allowed the optimization of a simple, fast and validated UPLC-PDA method which can be used to support the quality assessment of this herbal material. Keywords: Asteraceae, Eriodictyol, UPLC-PDA, Vernonanthura tweedieana

Ethnopharmacological relevance Ageratum conyzoides L. is a plant widely used in traditional medicine in tropical and subtropical regions of the world due to its anti-inflammatory, antinociceptive and antibacterial properties. Aim of the study To characterize the standardized extract of polymethoxyflavones (SEPAc) from the plant and evaluate its antinociceptive and anti-inflammatory effects. Materials and methods The SEPAc purified from the ethanol extract of the plant leaves was characterized by high resolution mass spectrometry and the methoxyflavones were quantified by a validated UPLC-PDA method. The antinociceptive and anti-inflammatory activities of the SEPAc were evaluated after oral administration on the acute nocifensive behavior of mice induced by formalin, prostaglandin E2 (PGE2) and proinflammatory cytokines (interleukin-1beta (IL-1β)) and tumor necrosis factor-alpha (TNF-α) in mice. Results Qualitative analyses revealed the presence of seven methoxyflavones in the SEPAc, also a simple UPLC-PDA method was developed and validated for the quantification of 5,6,7,3′,4′,5′-hexametoxyflavone; nobiletin; 5′-methoxynobiletin and eupalestin, major compounds in the extract. The SEPAc exhibited antinociceptive and anti-inflammatory activities in both formalin phases, with significant inhibition of the paw edema formation and significant reduction of the nocifensive response induced by an intraplantar injection of PGE2 and intrathecal injection of interleukin-1β. Conclusions The SEPAc exhibited significant antinociceptive and anti-inflammatory effects. These results provided scientific suggestion of its potential as a source of new medicines to treat inflammatory diseases, such rheumatoid arthritis.