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Erythrostemon yucatanensis (Greenm.) Gagnon & GP Lewis is a legume tree native to and widely distributed in southeast Mexico, where its branches are used in traditional medicine. An in vitro evaluation of the antiviral activity of extracts and fractions from the leaves, stem bark and roots against two strains of the AH1N1 influenza virus was performed, leading to the identification of bioactive compounds in this medicinal plant. In a cytopathic effect reduction assay, the fractions from the leaves and stem bark were the active elements at the co-treatment level. These were further fractionated based on their hemagglutination inhibition activity. The analysis of spectroscopy data identified a combination of phytosterols (ß-sitosterol, stigmasterol and campesterol) in the stem bark active fraction as the main anti-hemagglutinin binding components, while 5-hydroxy-2(2-hydroxy-3,4,5-trimethoxyphenyl)-7-metoxi-4H(chromen-4-ona), which was isolated from the leaf extracts, showed a weak inhibition of viral hemagglutinin. Time of addition experiments demonstrated that the mixture of sterols had a direct effect on viral particle infectivity at the co-treatment level (IC50 = 3.125 µg/mL). This effect was also observed in the virus plaque formation inhibition assay, where the mixture showed 90% inhibition in the first 20 min of co-treatment at the same concentration. Additionally, it was found using qRT-PCR that the NP copy number was reduced by 92.85% after 60 min of co-treatment. These results are the first report of components with anti-hemagglutinin binding activity in the genus Erythrostemon sp.

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Lignin is a technological bottleneck to convert polysaccharides into fermentable sugars, and different strategies of genetic-based metabolic engineering have been applied to improve biomass saccharification. Using maize seedlings grown hydroponically for 24 h, we conducted a quick non-transgenic approach with five enzyme inhibitors of the lignin and tricin pathways. Two compounds [3,4-(methylenedioxy)cinnamic acid: MDCA and 2,4-pyridinedicarboxylic acid: PDCA] revealed interesting findings on root growth, lignin composition, and saccharification. By inhibiting hydroxycinnamoyl-CoA ligase, a key enzyme of phenylpropanoid pathway, MDCA decreased the lignin content and improved saccharification, but it decreased root growth. By inhibiting flavone synthase, a key enzyme of tricin biosynthesis, PDCA decreased total lignin content and improved saccharification without affecting root growth. PDCA was three-fold more effective than MDCA, suggesting that controlling lignin biosynthesis with enzymatic inhibitors may be an attractive strategy to improve biomass saccharification.

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The Papilionoideae, which comprises 503 genera and approximately 14,000 species, is the largest and most diverse subfamily of the Fabaceae family. In this subfamily, the Crotalarieae, Genisteae, Podalyrieae, Thermopsideae, Sophoreae and Euchresteae tribes are closely related by micro and macromolecular features, thus forming the genistoid clade. This group combines well-known genera, whereas other genera lack phytochemical and chemotaxonomic studies. Thus, this work aimed to characterize the special metabolites in these genera in order to define the chemical profile, the micromolecular markers and the chemical diversity, as well as to evaluate the group evolutionary trends. Flavonoids and alkaloids were identified as chemosystematic markers for the studied tribes due to high occurrence number and structural diversity. Among flavonoids, the flavones and isoflavones predominated. Low protection indexes of flavonoid hydroxyls by O-glycosylation or O-methylation were observed, whereas C-prenylation and C-glycosylation were frequent, mainly at C-6 and C-8 positions. The flavone/flavonol ratio shows the predominance of the flavones. Quinolizidine and piperidine alkaloids were present in most genera. Pyrrolizidine alkaloids were found in a few genera from Thermopsideae, Genisteae and Crotalarieae, which suggests a mechanism of adaptive convergence. Cluster analysis allowed separation of genera for each tribe by chemical similarities. The micromolecular trends of protection of flavonoid hydroxyls and alkaloid oxidation indicate the genistoid clade is through evolutionary transition, which is consistent with its phylogenetic position in the Papilionoideae subfamily.

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Chrysin (5,7-dihydroxyflavone), a nutraceutical flavonoid present in diverse plants, has a backbone structure shared with the flavone backbone, with additional hydroxyl groups that confers its antioxidant properties and effects at the GABAA receptor complex. However, whether these effects are due to the hydroxyl groups is unknown. Here we report the effects of chrysin or the flavone backbone (1 mg/kg) in rats subjected to the elevated plus-maze and the locomotor activity test, as well as in the zebrafish evaluated in light/dark model. Chrysin, but not flavone, increased entries and time in the open arms of the elevated plus-maze, as well as time on white compartment of the light/dark model in zebrafish. These effects were comparable to diazepam, and were devoid of motor effects in both tests, as well as in the locomotor activity test. On the other hand, flavone decreased risk assessment in the light/dark test but increased rearing in the locomotor activity test in rats, suggesting effects threat information gathering; important species differences suggest new avenues of research. It is suggested that the specific effects of chrysin in relation to flavone include more of a mechanism of action in which in addition to its action at the GABAA/benzodiazepine receptor complex also could be involved its free radical scavenging abilities, which require specific research. Preprint: https://doi.org/10.1101/575514; Data and scripts:https://github.com/lanec-unifesspa/chrysin.

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Viral diseases transmitted by the female Aedes aegypti L. are considered a major public health problem. The aerial parts of Helicteres velutina K. Schum (Sterculiaceae) have demonstrated potential insecticidal and larvicidal activity against this vector. The objective of this research was to investigate the mechanisms of action involved in the larvicidal activity of this species. The cytotoxicity activity of H. velutina fractions and compounds of crude ethanolic extract of the aerial parts of this species was assessed by using fluorescence microscopy and propidium iodide staining. In addition, the production of nitric oxide (NO) and hemocyte recruitment were checked after different periods of exposure. The fluorescence microscopy revealed an increasing in larvae cell necrosis for the dichloromethane fraction, 7,4'-di-O-methyl-8-O-sulphate flavone and hexane fraction (15.4, 11.0, and 7.0%, respectively). The tiliroside did not show necrotic cells, which showed the same result as that seen in the negative control. The NO concentration in hemolymph after 24 h exposure was significantly greater for the dichloromethane fraction and the 7,4'-di-O-methyl-8-O-sulphate flavone (123.8 and 56.2 µM, respectively) when compared to the hexane fraction and tiliroside (10.8 and 8.3 µM, respectively). The presence of plasmocytes only in the dichloromethane fraction and 7,4'-di-O-methyl-8-O-sulphate flavone treatments suggest that these would be the hemocytes responsible for the highest NO production, acting as a defense agent. Our results showed that the larvicidal activity developed by H. velutina compounds is related to its hemocyte necrotizing activity and alteration in NO production.

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In this paper, we complement our previous study on the antiproliferative activity of Calea fruticosa (Asteraceae) by isolating the compounds apigenin-4',7-dimethyl ether (1), budlein A (2), quercetin (3), and cichoriin (4) from the plant's aerial parts. The antiproliferative activity of these compounds was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method against human tumor cell lines. Compound 3 displayed moderate antiproliferative activity in three cell lines (HCT-116, PC-3, and SF-295, with cell growth inhibition values of 72.97, 74.55, and 68.94%) and high antiproliferative activity (90.86%) in the HL-60 cell line. The in vitro sun protection factor (SPF) of the extracts and compound 4, with and without sunscreen, was determined by a spectrophotometric method. The ethanol extract exhibited the highest SPF (9.67) at a concentration of 0.100 mg/mL, while compound 4, isolated from this extract, showed a SPF of 13.79 at the same concentration. A relative increased efficacy of SPF was observed for the extracts and compound 4 when sunscreen was also used. Compound 4 has not been reported previously from any species within the genus Calea. Compounds 1-4 were obtained from this species for the first time.

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Cirsiliol is a flavone found in many Lamiaceae species with high cytotoxic activity against tumor cell lines. Although cirsiliol is being used in cancer therapy, its pharmacological potential is limited by its low solubility and bioavailability. In this paper, a cirsiliol-ß-cyclodextrin inclusion complex was developed in order to increase its solubility and bioavailability. The formation of inclusion complex was proved by scanning electron microscopy, Fourier-transform infrared spectroscopy (FTIR) and nuclear magnetic resonance (NMR) and solubility increment was verified through the ultraviolet-visible (UV-Vis) method. The cytotoxic effect against tumor cells (PC3, HCT-116 and HL-60 human cell lines, and S-180 murine cell line) and the antitumor activity in mice bearing sarcoma S-180 were also investigated. The inclusion complex was obtained with 71.45% of total recovery and solubility 2.1 times higher compared to the compound in its free form. This increment in solubility was responsible by a tumor growth inhibition potentiation (1.5 times greater compared to compound in its free form). In addition, this study showed that cirsiliol and its inclusion complex in ß-cyclodextrin have strong antitumor potential at low doses without promoting side effects commonly observed for conventional drugs as doxorubicin.

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The enzyme tyrosinase is involved in the biosynthesis of melanin and the enzymatic browning of fruits and vegetables, and therefore, its inhibitors have potential to treat hyperpigmentary disorders or to function as food antibrowning agents. The use of hydrazine monohydrate as a reagent to prepare chemically engineered extracts can lead to semisynthetic compounds that contain the portion N-N, a fragment rarely found in natural products and present in some tyrosinase inhibitors. Here, we report the tyrosinase inhibition screening of a series of chemically engineered extracts that are diversified by reaction with hydrazine. LC-MS was used to evaluate the change in composition produced by the reaction. Bioguided fractionation of the most active chemically engineered extract, prepared from Matricaria recutita L., led to the discovery of a pyrazole that inhibits tyrosinase with an IC50 value of 28.20 ± 1.13 µM. This compound was produced by a one-pot double chemical transformation of its natural precursor, which includes an unexpected selective removal of one -OH group.

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Helicteres velutina K. Schum (Sterculiaceae), a member of Malvaceae sensu lato, is a Brazilian endemic plant that has been used by the indigenous tribe Pankarare as an insect repellent. A previous study has reported the isolation of terpenoids, flavonoids and pheophytins, in addition to the larvicidal activity of crude H. velutina extracts derived from the aerial components (leaves, branches/twigs, and flowers). The present study reports the biomonitoring of the effects of fractions and isolated compounds derived from H. velutina against A. aegypti fourth instar larvae. A crude ethanol extract was submitted to liquid-liquid extraction with hexane, dichloromethane, ethyl acetate and n-butanol to obtain their respective fractions. Larvicidal evaluations of the fractions were performed, and the hexane and dichloromethane fractions exhibited greater activities than the other fractions, with LC50 (50% lethal concentration) values of 3.88 and 5.80 mg/mL, respectively. The phytochemical study of these fractions resulted in the isolation and identification of 17 compounds. The molecules were subjected to a virtual screening protocol, and five molecules presented potential larvicidal activity after analyses of their applicability domains. When molecular docking was analysed, only three of these compounds showed an ability to bind with sterol carrier protein-2 (1PZ4), a protein found in the larval intestine. The compounds tiliroside and 7,4'-di-O-methyl-8-O-sulphate flavone showed in vitro larvicidal activity, with LC50 values of 0.275 mg/mL after 72 h and 0.182 mg/mL after 24 h of exposure, respectively. This is the first study to demonstrate the larvicidal activity of sulphated flavonoids against A. aegypti. Our results showed that the presence of the OSO3H group attached to C-8 of the flavonoid was crucial to the larvicidal activity. This research supports the traditional use of H. velutina as an alternative insecticide for the control of A. aegypti, which is a vector for severe arboviruses, such as dengue and chikungunya.

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The steroselective oxa-Michael addition of the phenol moiety present in tyrosine and 3-iodotyrosine to different propargyl aldehydes delivered products with predominantly Z stereochemistry, as evidenced by X-ray crystallography analysis. When ethyl ropiolate was used as the propargyl ester source, the products were achieved with exclusively E stereochemistry with yields ranging from 17% to 91%. The oxa-Michael addition compounds served as substrates in the synthesis of 5- and 6-membered heterocyclic compounds. The atmosphere applied to the reaction medium directly influenced the formation of the products. When an inert atmosphere of nitrogen was applied, a 2-aryl-3-formyl-5-alanylbenzofuran core was selectively obtained via a Heck intramolecular reaction, while the reactions carried out under a carbon monoxide atmosphere led exclusively to 6-alanyl-2-arylflavone derivatives via reductive intramolecular acylation.

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In this research, the leaves of Lepechinia heteromorpha (Briq.) Epling, Lepechinia radula (Benth.) Epling and Lepechinia paniculata (Kunth) Epling have been collected in order to perform a phytochemical study. The first species was distilled to obtain a novel essential oil (EO), while the others were submitted to ethyl acetate extraction and secondary metabolite isolation. The chemical composition of the EO from L. heteromorpha has been investigated by Gas Chromatography-Mass Spectrometry (GC-MS) and Gas Chromatography with Retention Indices (GC(RI)), identifying 25 constituents. A major compound, (-)-ledol (21.2%), and a minor compound, (-)-caryophyllene oxide (1.0%), were isolated from the EO and their structures confirmed by Nuclear Magnetic Resonance (NMR) spectroscopy. Other major constituents of the EO were viridiflorene (27.3%), (E,E)-α-farnesene (1.4%), spirolepechinene and (E)-ß-caryophyllene (7.1% each), allo-aromadendrene (6.1%), camphor (1.7%), limonene (1.3%) and ß-phellandrene (4.6%). The enantiomeric composition of the EO monoterpene fraction was also studied, determining the enantiomeric excess and distribution of α-pinene, limonene, ß-phellandrene and camphor. The ethyl acetate extract of L. radula and L. paniculata were fractionated by column chromatography. Spathulenol, angustanoic acid E and 5-hydroxy-4',7-dimethoxy flavone were isolated from L. radula extract; ledol, guaiol and (-)-carnosol were found in L. paniculata.

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ABSTRACT The organic extracts from stems, roots and leaves of Tephrosia egregia Sandwith, Fabaceae, provided a new flavone, 5-hydroxy-8-(1",2"-epoxy-3"-hydroxy-3"-methylbutyl)-7-methoxyflavone (1), in addition to eleven known compounds: pongaflavone (2), praecansone B (3), 12a-hydroxyrotenone (4), praecansone A, 2',6'-dimethoxy-4',5'-(2",2"-dimethyl)-pyranochalcone, pongachalcone, maackiain, β-sistosterol and its glucoside, p-cumaric acid and cinnamic acid. The structures of all compounds were established on the basis of spectroscopic methods, mainly 1D and 2D NMR and HRESIMS, involving comparison with literature data. Cytotoxicity of compounds 1-4 was evaluated against AGP-01 (cancerous ascitic fluid), HCT-116 (colon adenocarcinoma), HL-60 (leukemia), PC-3 (prostate carcinoma), SF-295 (glioblastoma) and SKMEL 28 (melanoma) cell lines.

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The human tissue kallikreins (KLK1-KLK15) comprise a family of 15 serine peptidases detected in almost every tissue of the human body and that actively participate in many physiological and pathological events. Some kallikreins are involved in diseases for which no effective therapy is available, as for example, epithelial disorders, bacterial infections and in certain cancers metastatic processes. In recent years our group have made efforts to find inhibitors for all kallikreins, based on natural products and synthetic molecules, and all the inhibitors developed by our group presented a competitive mechanism of inhibition. Here we describe fukugetin, a natural product that presents a mixed-type mechanism of inhibition against KLK1 and KLK2. This type of inhibitor is gaining importance today, especially for the development of exosite-type inhibitors, which present potential to selectively inhibit the enzyme activity only against specific substrate.

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Lonchocarpus araripensis Benth. is largely distributed in the northeast region of Brazil. It is popularly known as 'sucupira'. Recent studies have shown that some species of Lonchocarpus have interesting pharmacological activities. In this study, we evaluated the antinociceptive effect of a flavone isolated from L. araripensis. The chemical examination resulted in the isolation of 3,6-dimethoxy-6″,6″-dimethyl-(7,8,2″,3″)-chromeneflavone (DDF). The structure of the compound was established by spectral analysis. Antinociceptive activity of DDF was evaluated by measuring nociception by acetic acid, formalin and hot plate tests. The rota rod test was used to evaluate motor coordination. The results demonstrated that DDF was able to prevent acetic-acid-writhing-induced nociception (p < 0.001) in mice. Furthermore, DDF produced a significant reduction of the nociceptive behaviour at the early and late phases of paw licking in the formalin test. Also, DDF produced an inhibition of the nociceptive behaviour during a hot-plate test. No alteration in motor coordination was observed. These results confirm the hypothesis that DDF reduces the nociceptive behaviour in mice, probably through central mechanisms, but without compromising the motor coordination of animals.

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Matrix assisted laser desorption ionization (MALDI) is a technique widely employed in the analysis of proteins and peptides, and nowadays it has also been applied to small molecules. There is little significant information regarding the in-source dissociation processes on MALDI for natural products. Twenty-six flavonoids (flavanones, flavones and flavonols) were analyzed by MALDI using different methods (with different matrices) and without matrix to comprehend the in-source reactions and establish good analysis methods for these compounds. Depending on the class, structure and the laser intensity applied, methoxylated flavonoid aglycones can eliminate methyl radicals (˙CH3) in the source, such as flavonols, but lithium 2,4-dihydroxybenzoate matrix suppresses the ˙CH3 eliminations and retro-Diels-Alder cleavages in the source. All of the flavonoid O-glycosides evaluated herein eliminated the sugar in source, even in the presence of the matrix, and its product radical ions ([M-H-sugar](-)˙) were observed in the negative mode. The flavone C-glycosides suffered intense dissociation, which was reduced by the addition of a matrix and the application of low laser intensity, mainly in the negative mode. Depending on the hydroxyl substituents, the [M-H-H](-)˙ ion was observed with variable relative intensity in the spectra.

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In this work, a bivalent RNA interference (RNAi) plant-transformation vector was constructed to silence both the flavanone 3-hydroxylase (F3H) gene and the flavone synthase II (GmFNSII) gene in soybean (Glycine max). Two further unit RNAi vectors were constructed for each of these two genes. RNAi-mediated suppression of these genes effectively regulated flavone and isoflavone production in hairy roots that arose from soybean cotyledons transformed with Agrobacterium rhizogenes ATCC15834. Notably, the bivalent RNAi vector had a significantly higher effect for increasing isoflavone production compared with the two unit RNAi vectors. The study highlighted molecular methods that could be used to enhance isoflavone production in soybean and demonstrated the challenges associated with such metabolic engineering for the production of plant natural products.

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Piper is a notable genus among Piperaceae due to their secondary metabolites such as lignans, amides, esters and long chain fatty acids used as anti-herbivore defenses with comparable effects of pyrethroids, that holds a promise in insect control, including malaria vectors such as Anopheles darlingi, the main vector in the North of Brazil. Methanolic extracts of Piper tuberculatum Jacq., Piperaceae, and P. alatabaccum Trel. & Yunck., Piperaceae, and some isolated compounds, i.e, 3,4,5-trimetoxy-dihydrocinamic acid, dihydropiplartine; piplartine, piplartine-dihydropiplartine and 5,5',7-trimetoxy-3',4'-metilenodioxiflavone were tested as larvicides against A. darlingi. The Lethal Concentrations (LC50 and LC90) of methanolic extracts were 194 and 333 ppm for P. tuberculatum and 235 and 401 ppm for P. alatabacum, respectively. Isolated compounds had lower LC values, e.g. the LC50 and LC90 of the piplartine-dihidropiplartine isolated from both plant species was 40 and 79 ppm, respectively.

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From the methanolic extract of the aerial parts of Ajuga chamaepitys (L.) Schreb., Lamiaceae, one of the Iranian medicinal plants, the phenylethanoid glycoside, acteoside, and two flavone glycosides, chrysoeriol 7-O-glucopyranoside (3'-methoxy-luteolin 7-O-glucopyranoside) and apigenin 7-O-rhamnopyranoside, were isolated by a combination of solid-phase extraction (SPE) and preparative reversed-phase high-performance liquid chromatography (prep-RP-HPLC) methods. Structures of the isolated compounds were elucidated by spectroscopic means. The free-radical-scavenging properties of the extracts, fractions and isolated compounds were determined by the 2,2-diphenyl-1-picryl-hydrazyl (DPPH) assay. While among the extracts, the MeOH extract showed the highest level of free-radical-scavenging activity (RC50 1.15 × 10-1 mg/mL), chrysoeriol 7-O-glucopyranoside was the most active (RC50 3.00 × 10-3 mg/mL) among the isolated compounds. The GC-MS and the GC-FID analyses revealed α-pinene (23.66%), β-pinene (9.33%), 1-octen-3-ol (9.72%), β-phellandrene (8.70%) and germacrene-D (7.92%) as the major components of the essential oils derived from the aerial parts of this plant. The presence of phenolic glycosides and the α- and β-pinene-rich essential oils in A. chamaepitys may provide some rationale for the traditional medicinal uses of this species in Iran.

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O ácido flavona-6-sulfônico, 4'-O-metil-5,7-diidroxi-flavona-6-sulfonato, conhecido como niruriflavona, e a saponina, ácido 3-O-[6'-O-4-hidroxibenzoil]-²-D-galactopiranosil-ursa-12-en-28-óico, foram isolados, respectivamente, de madeira e folhas de Licania arianeae. As estruturas foram estabelecidas através da análise de espectros de massas e RMN incluindo experimentos bidimensionais.

The flavone-6-sulfonic acid, 4'-metil-5,7-dihydroxy-flavone-6-sulfonic acid, known as niruriflavone, and the saponin 3-O-[6'-O-4-hydroxybenzoyl]-²-D-galactopyranosyl-ursa-12-en-28-oic acid were isolated, respectively, from wood and leaves of Licania arianeae. The structures were established from the NMR and mass spectra data analysis including two-dimensional NMR experiments.

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Preparative reversed-phase HPLC analysis of the methanol extract of the aerial parts of Chrozophora tinctoria (Euphorbiaceae) yielded five flavonoid glycosides, quercetin 3-O-rutinoside (1, rutin), acacetin 7-O-rutinoside (2), apigenin 7-O-b-D-[(6-p-coumaroyl)]-glucopyranoside (3), apigenin 7-O- b-D-glucopyranoside (4) and apigenin 7-O-b-D-[6-(3,4-dihydroxybenzoyl)]-glucopyranoside (named, chrozophorin, 5), the last one being a new natural product. The structures of these compounds were elucidated unambiguously by UV spectroscopic analyses using shift reagents, ESIMS, and 1D and 2D NMR spectroscopic techniques. The free-radical scavenging activity of the methanol extract (RC50 = 2.24 x 10-1 mg/mL) as well as the isolated compounds (1-5) (RC50 = 4.38 x 10-3, 2.26 x 10-2, 7.69 x 10-4, 8.71 x 10-3 and 3.19 x 10-4 mg/mL, respectively) were assessed by the DPPH assay.

Análise das partes aéreas de Chrozophora tinctoria (Euphorbiaceae) através de HPLC preparativa com coluna de fase reversa produziu cinco glicosídeos de flavonóides, quercetina 3-O-rutinosídeo (1, rutina), acacetina 7-O-rutinosídeo (2), apigenina 7-O-b-D-[(6-p-cumaroil)]-glicopiranosídeo (3), apigenina 7-O-b-D-glicopiranosídeo (4) e apigenina 7-O-b-D-[-(3,4-diidroxibenzoil)]-glicopiranosídeo (chamado crozoforina, 5), sendo o último um novo produto natural. As estruturas dessas substâncias foram inequivocamente elucidadas por análise de espectrofotometria de UV com o uso de reagentes de deslocamento, ESIMS, e técnicas de RMN 1D e 2D. A atividade de captura de radicais livres do extrato metanólico (RC50 = 2,24 x 10-1 mg/mL) bem como das substâncias isoladas (1-5) (RC50 = 4,38 x 10-3, 2,26 x 10-2, 7,69 x 10-4, 8,71 x 10-3 e 3,19 x 10-4 mg/mL, respectivamente) foram analisados pelo método DPPH.