Inhibitory effects of 190 compounds against SARS-CoV-2 Mpr o protein: Molecular docking interactions.

COVID-19 has caused many deaths since the first outbreak in 2019. The burden on healthcare systems around the world has been reduced by the success of vaccines. However, population adherence and the occurrence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants are still challenging tasks to be affronted. In addition, the newly approved drug presents some limitations in terms of side effects and drug interference, highlighting the importance of searching for new antiviral agents against SARS-CoV-2. The SARS-CoV-2 main protease (Mpr o ) represents a versatile target to search for new drug candidates due to its essential role in proteolytic activities responsible for the virus replication. In this work, a series of 190 compounds, composed of 27 natural ones and 163 synthetic compounds, were screened in vitro for their inhibitory effects against SARS-CoV-2 Mpro . Twenty-five compounds inhibited Mpro with inhibitory constant values (Ki ) between 23.2 and 241 µM. Among them, a thiosemicarbazone derivative was the most active compound. Molecular docking studies using Protein Data Bank ID 5RG1, 5RG2, and 5RG3 crystal structures of Mpro revealed important interactions identified as hydrophobic, hydrogen bonding and steric interactions with amino acid residues in the active site cavity. Overall, our findings indicate the described thiosemicarbazones as good candidates to be further explored to develop antiviral leads against SARS-CoV-2. Moreover, the studies showed the importance of careful evaluation of test results to detect and exclude false-positive findings.

Semisynthetic Sesquiterpene Lactones Generated by the Sensibility of Glaucolide B to Lewis and Brønsted-Lowry Acids and Bases: Cytotoxicity and Anti-Inflammatory Activities.

Sesquiterpene lactone (SL) subtypes including hirsutinolide and cadinanolide have a controversial genesis. Metabolites of these classes are either described as natural products or as artifacts produced via the influence of solvents, chromatographic mobile phases, and adsorbents used in phytochemical studies. Based on this divergence, and to better understand the sensibility of these metabolites, different pH conditions were used to prepare semisynthetic SLs and evaluate the anti-inflammatory and antiproliferative activities. Therefore, glaucolide B (1) was treated with various Brønsted-Lowry and Lewis acids and bases-the same approach was applied to some of its derivatives-allowing us to obtain 14 semisynthetic SL derivatives, 10 of which are hereby reported for the first time. Hirsutinolide derivatives 7a (CC50 = 5.0 µM; SI = 2.5) and 7b (CC50 = 11.2 µM; SI = 2.5) and the germacranolide derivative 8a (CC50 = 3.1 µM; SI = 3.0) revealed significant cytotoxic activity and selectivity against human melanoma SK-MEL-28 cells when compared with that against non-tumoral HUVEC cells. Additionally, compounds 7a and 7c.1 showed strongly reduced interleukin-6 (IL-6) and nitrite (NOx) release in pre-treated M1 macrophages J774A.1 when stimulated with lipopolysaccharide. Despite the fact that hirsutinolide and cadinanolide SLs may be produced via plant metabolism, this study shows that acidic and alkaline extraction and solid-phase purification processes can promote their formation.

Effect of Aryl-Cyclohexanones and their Derivatives on Macrophage Polarization In Vitro.

Macrophages are critical in both tissue homeostasis and inflammation, and shifts in their polarization have been indicated as pivotal for the resolution of inflammatory processes. Inflammation is a complex and necessary component of the immune response to stimuli that are harmful to host homeostasis and is regulated by cellular and molecular events that remain a source of ongoing investigation. Among the compounds studied that have potential against autoimmune and inflammatory diseases, cannabinoids are currently highlighted. In this work, nineteen aryl-cyclohexanones diesters and their derivatives were synthesized based on the aryl-cyclohexane skeleton of phytocannabinoids, such as cannabidiol (CBD), and were evaluated for their anti-inflammatory and macrophage polarization potential. The results showed that Compound 4 inhibited the production of nitric oxide in RAW 264.7 macrophages. Furthermore, it reduced the levels of pro-inflammatory cytokines IL-12p70, TNF-α, IFN-γ, MCP-1, and IL-6 while, at the same time, was able to increase the production of anti-inflammatory cytokines IL-4, IL-10, and IL-13. Compound 4 also reduced macrophage apoptosis, increased the expression of the CD206 (mannose receptor) and at the same time, decreased the expression of CD284 (TLR-4 receptor) on the surface of these cells. Finally, it increased the phagocytic capacity and inhibited the phosphorylation of the p65 of NF-kß. In conclusion, Compound 4, identified as diethyl-4-hydroxy-2-(4-methoxyphenyl)-4-methyl-6-oxocyclohexane-1-3-dicarboxylate, showed significant anti-inflammatory effect, while demonstrating the ability to transform phenotypically macrophages from the M1 phenotype (pro-inflammatory) to the M2 phenotype (anti-inflammatory). This led us to hypothesize that the main mechanism of anti-inflammatory effect of this molecule is linked to its immune modulation capacity.

Anti-inflammatory and anti-aggregating effects of rangpur in the first trimester of growth: ultra-performance liquid chromatography-electrospray mass spectrometry profile and quantification of hesperidin.

BACKGROUND: Citrus fruits are a rich source of valuable molecules, and their industrial processing produces bagasses, little explored to generate important by-products. These Citrus residues, including seeds and peels, also contain numerous pharmacologically important substances. To reduce the impact of these Citrus by-products, young, harvested fruits could be used as a functional supplemental food while another part is grown until maturity for industrial production. This study therefore aims to valorize rangpur (Citrus limonia) in the first 3 months of its growth by investigating and comparing its monthly chemical profiles using ultra-performance liquid chromatography-electrospray mass spectrometry (UPLC-ESI-MS) and its anti-inflammatory and antiplatelet activity. RESULTS: Extracts obtained from the fruits harvested in November, December, and January, 2017 and 2018 (L221117, L161217, and L160118) showed different UPLC-ESI-MS profiles. Twenty-five of the 26 detected metabolites were identified as cyclitol, pyrrolidine betaine, aryl propanoyl esters, chlorogenic acids, flavonoids, coumarins, and limonoids. Quantification studies indicated an increased concentration of hesperidin from the younger fruits to the older fruits of the series. L160118 reduced nitrogen oxide (NOx), tumor necrosis factor alpha (TNF-α), and interleukin 6 (IL-6) levels more than other extracts. Their activity followed the same trends as the hesperidin concentration in each fruit. In contrast, the most promising antiplatelet activity was observed with the extracts from the two youngest fruits. This suggests combined effects of the chemical components found in these fruits' extracts. CONCLUSION: The extracts obtained from these young fruits showed considerable anti-inflammatory and antiplatelet activity. Overall, young rangpur could be used as raw material to produce functional foods without producing any waste. © 2022 Society of Chemical Industry.

Discovery of Spilanthol Endoperoxide as a Redox Natural Compound Active against Mammalian Prx3 and Chlamydia trachomatis Infection.

Chlamydia trachomatis (Ct) is a bacterial intracellular pathogen responsible for a plethora of diseases ranging from blindness to pelvic inflammatory diseases and cervical cancer. Although this disease is effectively treated with antibiotics, concerns for development of resistance prompt the need for new low-cost treatments. Here we report the activity of spilanthol (SPL), a natural compound with demonstrated anti-inflammatory properties, against Ct infections. Using chemical probes selective for imaging mitochondrial protein sulfenylation and complementary assays, we identify an increase in mitochondrial oxidative state by SPL as the underlying mechanism leading to disruption of host cell F-actin cytoskeletal organization and inhibition of chlamydial infection. The peroxidation product of SPL (SPL endoperoxide, SPLE), envisioned to be the active compound in the cellular milieu, was chemically synthesized and showed more potent anti-chlamydial activity. Comparison of SPL and SPLE reactivity with mammalian peroxiredoxins, demonstrated preferred reactivity of SPLE with Prx3, and virtual lack of SPL reaction with any of the reduced Prx isoforms investigated. Cumulatively, these findings support the function of SPL as a pro-drug, which is converted to SPLE in the cellular milieu leading to inhibition of Prx3, increased mitochondrial oxidation and disruption of F-actin network, and inhibition of Ct infection.

Search for Antimicrobial Activity Among Fifty-Two Natural and Synthetic Compounds Identifies Anthraquinone and Polyacetylene Classes That Inhibit Mycobacterium tuberculosis.

Drug-resistant tuberculosis threatens to undermine global control programs by limiting treatment options. New antimicrobial drugs are required, derived from new chemical classes. Natural products offer extensive chemical diversity and inspiration for synthetic chemistry. Here, we isolate, synthesize and test a library of 52 natural and synthetic compounds for activity against Mycobacterium tuberculosis. We identify seven compounds as antimycobacterial, including the natural products isobavachalcone and isoneorautenol, and a synthetic chromene. The plant-derived secondary metabolite damnacanthal was the most active compound with the lowest minimum inhibitory concentration of 13.07 µg/mL and a favorable selectivity index value. Three synthetic polyacetylene compounds demonstrated antimycobacterial activity, with the lowest MIC of 17.88 µg/mL. These results suggest new avenues for drug discovery, expanding antimicrobial compound chemistries to novel anthraquinone and polyacetylene scaffolds in the search for new drugs to treat drug-resistant bacterial diseases.

NOx-, IL-1ß-, TNF-α-, and IL-6-Inhibiting Effects and Trypanocidal Activity of Banana (Musa acuminata) Bracts and Flowers: UPLC-HRESI-MS Detection of Phenylpropanoid Sucrose Esters.

Banana inflorescences are a byproduct of banana cultivation consumed in various regions of Brazil as a non-conventional food. This byproduct represents an alternative food supply that can contribute to the resolution of nutritional problems and hunger. This product is also used in Asia as a traditional remedy for the treatment of various illnesses such as bronchitis and dysentery. However, there is a lack of chemical and pharmacological data to support its consumption as a functional food. Therefore, this work aimed to study the anti-inflammatory action of Musa acuminata blossom by quantifying the cytokine levels (NOx, IL-1ß, TNF-α, and IL-6) in peritoneal neutrophils, and to study its antiparasitic activities using the intracellular forms of T. cruzi, L. amazonensis, and L. infantum. This work also aimed to establish the chemical profile of the inflorescence using UPLC-ESI-MS analysis. Flowers and the crude bract extracts were partitioned in dichloromethane and n-butanol to afford four fractions (FDCM, FNBU, BDCM, and BNBU). FDCM showed moderate trypanocidal activity and promising anti-inflammatory properties by inhibiting IL-1ß, TNF-α, and IL-6. BDCM significantly inhibited the secretion of TNF-α, while BNBU was active against IL-6 and NOx. LCMS data of these fractions revealed an unprecedented presence of arylpropanoid sucroses alongside flavonoids, triterpenes, benzofurans, stilbenes, and iridoids. The obtained results revealed that banana inflorescences could be used as an anti-inflammatory food ingredient to control inflammatory diseases.

ESI-QTof-MS characterization of hirsutinolide and glaucolide sesquiterpene lactones: Fragmentation mechanisms and differentiation based on Na+ /H+ adducts interactions in complex mixture.

Sesquiterpene lactones (SL) have been reported with various biological effects. Among the described SL skeletons, hirsutinolide and glaucolide have not been extensively studied by mass spectrometry (MS), especially how to distinguish them in organic matrices. Thus, this paper reports (1) a strategy of their differentiation based on MS behavior during the ionization and (2) a proposal of the fragmentation pattern for both SL-subtypes. ESI(+)-HRMS data of four isolated SL (hirsutinolides 1 and 3; glaucolides 2 and 4) were recorded by direct and UPLC water-sample combined injections. These analyses revealed that hirsutinolides and glaucolides formed [M+Na]+ ion during the operation of the direct MS injection, and ([M+Na]+ and [M+H-H2 O]+ ) and [M+H]+ ions were respectively observed for hirsutinolides and glaucolides during the operation of combined UPLC water and sample MS injection. Computational simulations showed that the complex hirsutinolide (1)-Na+ formed with a lower preparation energy compared with the complex glaucolide (2)-Na+ . However, despite their different behavior during the ionization process, ESI(+)-HRMS/MS analyses of 1-4 gave similar fragmentation patterns at m/z 277, 259, 241, and 231 that can be used as diagnostic ions for both skeletons. Moreover, the differentiation strategy based on the nature of the complex SL-adducts and their MS/MS fragmentation pattern were successfully applied for the chemical characterization of the extract from Vernonanthura tweedieana using UPLC-ESI-HRMS/MS. Among the characterized metabolites, SL with hirsutinolide and glaucolide skeletons showed the aforementioned diagnostic fragments and an ionization behavior that was similar to those observed during the water-sample combined injection.

Correction: The value of universally available raw NMR data for transparency, reproducibility, and integrity in natural product research.

Correction for 'The value of universally available raw NMR data for transparency, reproducibility, and integrity in natural product research' by James B. McAlpine et al., Nat. Prod. Rep., 2018, DOI: .

The value of universally available raw NMR data for transparency, reproducibility, and integrity in natural product research.

Covering: up to 2018With contributions from the global natural product (NP) research community, and continuing the Raw Data Initiative, this review collects a comprehensive demonstration of the immense scientific value of disseminating raw nuclear magnetic resonance (NMR) data, independently of, and in parallel with, classical publishing outlets. A comprehensive compilation of historic to present-day cases as well as contemporary and future applications show that addressing the urgent need for a repository of publicly accessible raw NMR data has the potential to transform natural products (NPs) and associated fields of chemical and biomedical research. The call for advancing open sharing mechanisms for raw data is intended to enhance the transparency of experimental protocols, augment the reproducibility of reported outcomes, including biological studies, become a regular component of responsible research, and thereby enrich the integrity of NP research and related fields.

Prospecting for cytotoxic and antiprotozoal 4-aryl-4H-chromenes and 10-aryldihydropyrano[2,3-f]chromenes.

Different studies reported that genetic predisposition or metabolic dysfunction are the risk factors for cancer. Infectious parasitic diseases were listed among factors that predispose to cancer. Because of the resemblance between the life cycle of cancer cells and some parasites, this study aimed to prepare pyran derivatives with cytotoxic and antiprotozoal potencies. Therefore, 7 chromenes, 10 pyranocoumarins, and an unexpected intermediate were obtained from a multi-reagent one-pot reaction. These compounds were evaluated for their cytotoxicity on sensitive and resistant leukemia cancer cells lines and against two protozoan parasites, namely Trypanosoma cruzi and Leishmania amazonensis amastigote. Promising cytotoxicity (IC50 values of less than 1 µM) was obtained for two of the synthetic products (12 and 15). Compound 12 induced apoptosis and cell cycle arrest in CCRF-CEM leukemia cells in G0/G1 while compound 15 and doxorubicin induced apoptosis and arrest in the S and G2/M phases. Ten of these products showed trypanocidal activity, while only five of them were weakly active on L. amazonensis. Three of the obtained pyrans showed significant cytotoxicity and antitrypanocidal activity, simultaneously. Nevertheless, all antiparasitic compounds revealed potency with low selectivity toward THP-1 cells used as host.

A LC-MS validated method for quantification of rosmarinic acid and sesquiterpene lactones in Hedyosmum brasiliense.

The leaves of the aromatic neotropical shrub Hedyosmum brasiliense are employed popularly as a sedative, aphrodisiac and as a substitute for green tea. Sesquiterpene lactones and phenolic compounds were characterized as the main compounds of its aqueous extract, and some biological investigation demonstrated its anxiolytic, antidepressant and hypnotic effects. The quantification of podoandin, onoseriolide and rosmarinic acid in its infused tea was achieved by means of ultra high performance liquid chromatography coupled to a electronspray ionization interface and a high resolution mass detector. Quantification of the analytes was performed employing the areas of the extracted ion chromatograms of the analytes, negative ion mode for rosmarinic acid (1) and positive mode for podoandin (2) and onoseriolide (3). The method was validated by evaluating specificity, linearity, precision and accuracy and has been found to be sensitive, precise and accurate. When applied to analyze the hot water infusion extract of H. brasiliense, compounds 1, 2 and 3 were obtained to be 188 ± 1.45 mg/g, 1.9 ± 0.15 mg/g and 1.7 ± 0.02 mg/g of extract, respectively. The H. brasiliense tea was found to be a good source of the rosmarinic acid, also widely employed in the cosmetic industry.

UPLC-ESI-QTOF-MS2 characterisation of Cola nitida resin fractions with inhibitory effects on NO and TNF-α released by LPS-activated J774 macrophage and on Trypanosoma cruzi and Leishmania amazonensis.

INTRODUCTION: The resin of Cola nitida is used in western Cameroon as incense for spiritual protection and during ritual ceremonies. This plant secretion has never been investigated although previous chemical and biological studies on other resins have drawn many attentions. OBJECTIVE: The resin fractions which revealed inhibitory effect on nitric oxide (NO) and tumour necrosis factor alpha (TNF-α) released by lipopolysaccharide (LPS)-activated J774 macrophage as well as on intracellular forms of Leishmania amazonensis and Trypanosoma cruzi amastigote were chemically characterised. Moreover, their antiparasitic activities were compared to those of semi-synthetic triterpenes. METHODOLOGY: The anti-inflammatory activity was evaluated by measuring the nitrite production and the TNF-α concentration in the supernatants of LPS-activated macrophages by antigen capture enzyme-linked immunosorbent assay. Moreover, the antiparasitic assay was performed by infecting the host cells (THP-1) in a ratio parasite/cell 10:1 (L. amazonensis) and 2:1 (T. cruzi) and then exposed to the samples. The resin was separated in vacuo by liquid chromatography because of its sticky behaviour and the chemical profiles of the obtained fractions (F1-F4) were established by dereplication based on UPLC-ESI-MS2 data while semi-synthetic triterpenes were prepared from α-amyrin by oxidation reactions. RESULTS: Fractions F1-F4 inhibited NO and TNF-α almost similarly. However, only F1, F3 and F4 showed promising antiparasitic activities while F2 was moderately active against both parasites. Hence, F1-F4 were exclusively composed of pentacyclic triterpenes bearing oleanane and ursane skeletons. Semi-synthetic compounds revealed no to moderate antiparasitic activity compared to the fractions. CONCLUSION: Although it will be difficult to prove the interaction resin-spirit, interesting bioactivities were found in the resin fractions.

Cytotoxicity mechanisms in melanoma cells and UPLC-QTOF/MS2 chemical characterization of two Brazilian stingless bee propolis: Uncommon presence of piperidinic alkaloids.

The present study characterized propolis extracts produced by Scaptotrigona bipunctata (Tubuna) and Melipona quadrifasciata (Mandaçaia) 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. Mandaçaia'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 Mandaçaia's propolis extract induced cell cycle arrest in G2/M.

A new polyacetylene glucoside from Vernonia scorpioides and its potential antihyperglycemic effect.

Natural polyacetylene compounds have been found mainly in seven botanical families and remain underexplored and understudied, despite its inherent chemical and biological reactivity, due to the presence of conjugated triple bonds. Some polyacetylene glucosides have been found to stimulate glucose uptake in C5BL/ks-db/db obese diabetic mice, and since polyacetylene glucosides previously found in Vernonia scorpioides showed little to none cytotoxicity, in this study the antihyperglycemic potential of a new V. scorpioides polyacetylene glucoside has been accessed in order to shine a new light on the biological activity of this unique scaffold. For the isolation of this new compound an optimized method of Centrifugal Partition Chromatography (CPC) is for the first time described together with its X-ray data. The results demonstrate that 3,4-dihydrovernoniyne-4-O-ß-glucoside has significant effect on glycaemia at low dose 0.5 mg/kg, and pointing that the anti-hyperglycemic effect may be due in part to the inhibition of intestinal disaccharidases.

Synthesis of lupeol derivatives and their antileishmanial and antitrypanosomal activities.

The natural product lupeol 1 was isolated from aerial parts of Vernonia scorpioides with satisfactory yield, which made it viable to be used as starting material in semisynthetic approach. Ten lupeol derivatives 2-11 were prepared by classical procedures. Including, five new esters derivatives 7-11, which were obtained by structural modifications in the isopropylidene fragment. All semisynthetic compounds and lupeol 1-11 were confirmed by 1H NMR, 13C NMR and HRMS. Their antiprotozoal activity was evaluated in vitro against L. amazonensis and T. cruzi. Derivative 6 showed the best antitrypanosomal activity (IC50 = 12.48 µg/mL) and the lowest cytotoxic derivative (CC50 = 161.50 µg/mL). The mechanism of action of the most active derivatives (4, 6 and 11) is not dependent from the enzyme trypanothione reductase.

Synthesis and in vitro evaluation of Ca2+ channel blockers 1,4-dihydropyridines analogues against Trypanosoma cruzi and Leishmania amazonensis: SAR analysis.

Drugs containing the1,4-dihydropyridine (DHP) core have recently attracted attention concerning their antiparasitic effect against various species of Leishmania and Trypanosoma. This approach named drugs repositioning led to interesting results, which have prompted us to prepare 21 DHP's analogues. The 1,4-DHP scaffold was decorated with different function groups at tree points including the nitrogen atom (NH and N-phenyl), the aryl group attached to C-4 (various substituted aryl residues) and the carbon atoms 2 and 6 (bearing Ph or Me groups). Moreover, the products were evaluated for their cytotoxicity on three cancer and a non-tumoral cell lines. Only 6 of them were antiproliferative and their weak effect (CC50 comprised between 27 and 98µM) suggested these DHPs as good candidates against the intracellular amastigote forms of L. amazonensis and T. cruzi. L. amazonensis was sensitive to DHPs 5, 11 and 15 (IC50 values at 15.11, 45.70 and 53.13µM, respectively) while 12 of them displayed significant to moderate trypanocidal activities against T. cruzi. The best trypanocidal activities were obtained with compounds 2, 18 and 21 showing IC50 values at 4.95, 5.44, and 6.64µM, respectively. A part of the N-phenylated DHPs showed a better selectivity than their NH analogues towards THP-1 cells. 4-Chlorophenyl, 4-nitrophenyl and 3-nitrophenyl residues attached to the carbon atom 4 turned to be important sub-structures for the antitrypanosomal activity.

ESI-MS2 and Anti-inflammatory Studies of Cyclopropanic Triterpenes. UPLC-ESI-MS and MS2 Search of Related Metabolites from Donella ubanguiensis.

INTRODUCTION: Triterpenes are one of the largest secondary metabolites groups spread in the plant kingdom with various skeletons. These metabolites have showed various bioactivities including anti-inflammatory activity. OBJECTIVE: The study aims to explore the mass spectrometry fragmentation of donellanic acids A-C (DA A-C), three compounds identified from Donella ubanguiensis; in addition, the fragmentation behaviour of these metabolites will serve as a fingerprint to search and characterise triterpenes congeners in fruits, bark and wood crude extracts of D. ubanguiensis. This work was prompted by the anti-inflammatory activity on leukocyte migration, exudate concentrations and myeloperoxidase activity obtained for DA A-B. METHODOLOGY: The bioactivity was performed on mouse model of pleurisy induced by carrageenan and the parameters were analysed by veterinarian automated cell counter and colorimetric assays. While the tandem mass analyses of DA A-C were carried out by a direct infusion ESI-QTOF-MS/MS, the extracts were studied by UPLC-ESI-QTOF-MS and UPLC-ESI-QTOF-MS/MS. RESULTS: DA A displayed interesting anti-inflammatory activity by inhibiting leukocyte migration, exudate concentrations and myeloperoxidase activity (p < 0.05) while DA B was weakly active (p > 0.05). Moreover, the diagnostic of the MS2 behaviour of DA A-C in conjunction with the chromatograms and the obtained MS2 data of the crude extract led to the characterisation of three cyclopropane triterpenes (T1-T3) and six saponins (T4-T9) from the fruits, the bark, and the wood extracts. CONCLUSIONS: Donella species deserve more investigation since metabolites related to the anti-inflammatory compound (DA A) could be identified. Copyright © 2016 John Wiley & Sons, Ltd.

Simultaneous identification and quantification of polymethoxyflavones, coumarin and phenolic acids in Ageratum conyzoides by UPLC-ESI-QToF-MS and UPLC-PDA.

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.

Antinociceptive and anti-inflammatory activities of standardized extract of polymethoxyflavones from Ageratum conyzoides.

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-1ß)) 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-1ß. 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.