Characterization of the fragmentation mechanisms in electrospray ionization tandem mass spectrometry of chloroquinoline derivatives with larvicidal activity against Aedes aegypti.

RATIONALE: 4,7-Dichloroquinoline (DCQ) represents a group of synthetic molecules inspired by natural products with important roles in biological and biomedical areas. This work aimed to characterize DCQ and its derivatives by high-resolution electrospray ionization (ESI) mass spectrometry and tandem mass spectrometry (ESI-MS/MS), supported by theoretical calculations. Biological assays were carried out with DCQ and its derivatives to determine LC50 values against Aedes aegypti larvae. METHODS: Five DCQ derivatives were synthesized by using previously described protocols. ESI-MS/MS analyses were carried out with a quadrupole/time-of-flight and ion-trap instrument. The proposed gas-phase protonation sites and fragmentation were supported by density functional theory calculations. The larvicidal tests were performed with the Ae. aegypti Rockefeller strain, and the LC50 values were determined by employing five test concentrations. Larval mortality was determined after treatment for 48 h. RESULTS: DCQ bromides or aldehydes (C-3 or C-8 positions), as well as the trimethylsilyl derivative (C-3 position), were prepared. Detailed ESI-MS/MS data revealed heteroatom elimination through an exception to the even-electron rule, to originate open-shell species. Computational studies were used to define the protonation sites and fragmentation pathways. High activity of DCQ and its derivatives against Ae. aegypti larvae was demonstrated. CONCLUSION: Our results provided a well-founded characterization of the fragmentation reactions of DCQ and its derivatives, which can be useful for complementary studies of the development of a larvicidal product against Ae. aegypti.

Unveiling new myrsinoic acids and AChE ligands from Myrsine guianensis (Aubl.) Kuntze.

Molecular dereplication and drug-like discovery are important tools for exploring the chemical profile of metabolites in a complex mixture. In order to establish a workflow for discovering novel acetylcholinesterase (AChE) ligands, we performed the chemical study of Myrsine guianensis (Aubl.) Kuntze (Primulaceae). To carry out the bioprospection, nine extracts were obtained from different parts of the plant. Through the dereplication approaches, seventeen metabolites were annotated. In order to confirm the putative inferences, a HPLC preparative method was developed to isolate three known myrsinoic acids, A(1), B(2) and C(3). Along with, we are reporting the obtention of two new congeners, G(5) and H(6), which their structures were elucidated by NMR and HRMS data. Besides that, two extracts were submitted to affinity assays to accelerate the discovery of AChE ligands. Desorbates were analyzed through LC-HRMS for calculating the affinity ratio (AR). Thus, (1) presented AR = 4.59, therefore was considered a potential ligand.

Limonoids and insecticidal activity on Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) of Trichilia catigua A. Juss. (Meliaceae).

The aim of the study was to evaluate the effects of the seeds, exocarp and aril extracts from Trichilia catigua A. Juss. (Meliaceae) against Spodoptera frugiperda and present the phytochemical study carried out with the aril extract of T. catigua. Limonoids were isolated from the aril of T. catigua through chromatographic techniques and their structures were proposed by spectroscopic analysis and comparison with literature data. The effects of the seeds, exocarp and aril extracts from T. catigua against S. frugiperda were evaluated considering as parameters the duration and mortality of the larval phase, in addition to the pupal weight. Phytochemical investigation of the aril extracts of T. catigua has led to the identification of the limonoids 6α-O-acetyl-7-deacetyl-14,15-dihydro-15-oxo-nimocinol (1), cedrelone (2) and 6α-O-acetyl-7-deacetylnimocinol (3). The hexane and CH2Cl2 extracts of the aril showed a high rate of larval mortality (100 and 90%, respectively). In addition, a prolongation of larval phase and a reduction in the pupal weight were observed for insects treated with hexane, CH2Cl2 and methanol extracts of seeds and with CH2Cl2 extract of exocarp of T. catigua.

Anacardic Acid Derivatives Affect the in Vitro Reactions of Photosynthesis.

The dichloromethane extract of the cashew nuts from Anacardium occidentale was fractionated by rotation locular countercurrent chromatography aimed at discovering metabolites that could be useful as new models for photosynthesis inhibitors. The chemical fractionation afforded a complex mixture of anacardic acids, which upon catalytic hydrogenation yielded anacardic acid (1). Methylation of 1 via reaction with diazomethane afforded an ester 2. Both compounds were evaluated using polarographic approaches and fluorescence studies of chlorophyll a (ChL a). The in vitro assays informed the decision for the classification of 1 and 2 as Hill reaction inhibitors. Besides that, 1 inhibited the donor side of the PSII, while 2 acted as an energy transfer inhibitor. Therefore, this study is important for the development of herbicides.

Combination of GC-MS Molecular Networking and Larvicidal Effect against Aedes aegypti for the Discovery of Bioactive Substances in Commercial Essential Oils.

Dengue is a neglected disease, present mainly in tropical countries, with more than 5.2 million cases reported in 2019. Vector control remains the most effective protective measure against dengue and other arboviruses. Synthetic insecticides based on organophosphates, pyrethroids, carbamates, neonicotinoids and oxadiazines are unattractive due to their high degree of toxicity to humans, animals and the environment. Conversely, natural-product-based larvicides/insecticides, such as essential oils, present high efficiency, low environmental toxicity and can be easily scaled up for industrial processes. However, essential oils are highly complex and require modern analytical and computational approaches to streamline the identification of bioactive substances. This study combined the GC-MS spectral similarity network approach with larvicidal assays as a new strategy for the discovery of potential bioactive substances in complex biological samples, enabling the systematic and simultaneous annotation of substances in 20 essential oils through LC50 larvicidal assays. This strategy allowed rapid intuitive discovery of distribution patterns between families and metabolic classes in clusters, and the prediction of larvicidal properties of acyclic monoterpene derivatives, including citral, neral, citronellal and citronellol, and their acetate forms (LC50 < 50 µg/mL).

Chemometrics applied to 1H NMR and UV-Vis spectroscopy as a way to evaluate solid-liquid extraction of leaves of artichoke.

Artichoke has several biological actions, which are related to the synergistic action of its bioactive compounds. Solid-liquid extraction influence the type and quantity of compounds extracted and, consequently, the biological activity of the plant extract. This study aims to investigate which extraction method (maceration, infusion, or Soxhlet) and which green solvent (ethanol or acetone) would be more suitable to obtain bioactive artichoke extracts. All solid-liquid procedures were carried out in triplicate, using 3.0 g of artichoke leaves and 200 mL of solvent. After drying, samples were analyzed by UV-Vis and 1H NMR. Chemometrics was applied to spectroscopic data, and the PCA analysis showed that they were specially separated according to the solvent extractor. The sesquiterpene lactone cynaropicrin was identified as the major compound of the extracts, and this allowed us to conclude that the best solid-liquid procedure was Soxhlet, using ethanol as solvent.

Chemodiversity and Anti-Leukemia Effect of Metabolites from Penicillium setosum CMLD 18.

Penicillium setosum represents a Penicillium species recently described, with little up-to-date information about its metabolic and biological potential. Due to this scenario, we performed chemical and biological studies of P. setosum CMLD18, a strain isolated from Swinglea glutinosa (Rutaceae). HRMS-MS guided dereplication strategies and anti-leukemia assays conducted the isolation and characterization of six compounds after several chromatographic procedures: 2-chloroemodic acid (2), 2-chloro-1,3,8-trihydroxy-6- (hydroxymethyl)-anthraquinone (7), 7-chloroemodin (8), bisdethiobis(methylthio)acetylaranotine (9), fellutanine C (10), and 4-methyl-5,6-diihydro-2H-pyran-2-one (15). From the assayed metabolites, (10) induced cellular death against Kasumi-1, a human leukemia cell line, as well as good selectivity for it, displaying promising cytotoxic activity. Here, the correct NMR signal assignments for (9) are also described. Therefore, this work highlights more detailed knowledge about the P. setosum chemical profile as well as its biological potential, offering prospects for obtaining natural products with anti-leukemia capabilities.

Insecticidal activity of copper (II) complexes with flavanone derivatives.

In this work, we describe the insecticidal activity against Spodoptera frugiperda of flavanone derivatives (naringin, naringenin and hesperidin) complexed to copper (II). For the assays, the compounds were incorporated into an artificial diet and offered to S. frugiperda during its larval period. The duration of larval and pupal phases, the weight of pupae and the percentage of dead insects at the end of the larval phase were evaluated. Among the tested compounds, the free flavanones 1 and 3 and the complexes 4 and 6 showed a shortening of the larval phase when compared to the control. Complex 9 showed the highest toxicity, with 96.66% larval mortality. This is the first report on the insecticidal activity of the Cu2+ complexes of the flavanones naringin, naringenin and hesperidin.[Formula: see text].

Liquid Fungal Cocultivation as a Strategy to Access Bioactive Metabolites.

Fungi are a rich source of bioactive compounds. Fungal cocultivation is a method of potentiating chemical interactions and, consequently, increasing bioactive molecule production. In this study, we evaluated the bactericidal, antiprotozoal, and cathepsin V inhibition activities of extracts from axenic cultures of 6 fungi (Fusarium guttiforme, Pestalotiopsis diospyri, Phoma caricae-papayae, Colletotrichum horii, Phytophthora palmivora, and C. gloeosporioides) that infest tropical fruits and 57 extracts obtained by their cocultivation. Our results reveal that fungal cocultivation enhances the biological activity of the samples, since all extracts that were active on Gram-positive bacteria, Gram-negative bacteria, Trypanosoma cruzi, and Leishmania infantum were obtained from cocultivation. Bacterial growth is either totally or partially inhibited by 46% of the extracts. Two extracts containing mainly fusaric and 9,10-dehydrofusaric acids were particularly active. The presence of the fungus F. guttiforme in co-cultures that give rise to extracts with the highest activities against L. infantum. An axenic culture gave rise to the most active extract for the inhibition of cathepsin V; however, other coculture extracts also exhibited activity toward this biological target. Therefore, the results of the biological activities indicate that fungal cocultivation increased the biological potential of samples, likely due to the hostile and competitive environment that pushes microorganisms to produce substances important for defense and allows access to metabolic routes then silenced in milder cultivation conditions.

[10]-Gingerol improves doxorubicin anticancer activity and decreases its side effects in triple negative breast cancer models.

PURPOSE: Although doxorubicin is widely used to treat cancer, severe side effects limit its clinical use. Combination of standard chemotherapy with natural products can increase the efficacy and attenuate the side effects of current therapies. Here we studied the anticancer effects of a combined regimen comprising doxorubicin and [10]-gingerol against triple-negative breast cancer, which does not respond to hormonal or targeted therapies. METHODS: Cytotoxicity was evaluated by MTT assay, cell cycle progression and apoptosis were analyzed by flow cytometry and signaling pathways were analyzed by Western blotting in human and murine triple negative breast cancer cell systems. The anticancer/antimetastatic and toxic effects of the combined regimen was evaluated using syngeneic and xenograft orthotopic models. RESULTS: The combination of doxorubicin and [10]-gingerol significantly increased the number of apoptotic cells, compared to each compound alone. In 4T1Br4 cells, the combined regimen was the only condition able to increase the levels of active caspase 3 and γH2AX and to decrease the level of Cdk-6 cyclin. In vivo, doxorubicin (3 mg/Kg, D3) and [10]-gingerol (10 mg/Kg, G10) resulted in a significant reduction in the volume of primary tumors and a decrease in the number of circulating tumor cells (CTCs). Interestingly, only the combined regimen led to decreased tumor burdens to distant organs (i.e., metastasis) and reduced chemotherapy-induced weight loss and hepatotoxicity in tumor-bearing animals. Likewise, in a xenograft model, only the combined regimen was effective in significantly reducing the primary tumor volume and the prevalence of CTCs. CONCLUSIONS: Our data indicate that [10]-gingerol has potential to be used as a neoadjuvant or in combined therapy with doxorubicin, to improve its anticancer activity.

Plant Natural Products for the Control of Aedes aegypti: The Main Vector of Important Arboviruses.

The mosquito species Aedes aegypti is one of the main vectors of arboviruses, including dengue, Zika and chikungunya. Considering the deficiency or absence of vaccines to prevent these diseases, vector control remains an important strategy. The use of plant natural product-based insecticides constitutes an alternative to chemical insecticides as they are degraded more easily and are less harmful to the environment, not to mention their lower toxicity to non-target insects. This review details plant species and their secondary metabolites that have demonstrated insecticidal properties (ovicidal, larvicidal, pupicidal, adulticidal, repellent and ovipositional effects) against the mosquito, together with their mechanisms of action. In particular, essential oils and some of their chemical constituents such as terpenoids and phenylpropanoids offer distinct advantages. Thiophenes, amides and alkaloids also possess high larvicidal and adulticidal activities, adding to the wealth of plant natural products with potential in vector control applications.

Mycotoxins from Fusarium proliferatum: new inhibitors of papain-like cysteine proteases.

Papain-like cysteine proteases (PLCPs) in plants are essential to prevent phytopathogen invasion. In order to search for cysteine protease inhibitors and to investigate compounds that could be associated to pineapple Fusarium disease, a chemistry investigation was performed on Fusarium proliferatum isolated from Ananas comosus (pineapple) and cultivated in Czapek medium. From F. proliferatum extracts, nine secondary metabolites were isolated and characterized by nuclear magnetic resonance spectroscopy and mass spectrometry experiments: beauvericin (1), fusaric acid (2), N-ethyl-3-phenylacetamide (3), N-acetyltryptamine (4), cyclo(L-Val-L-Pro) cyclodipeptide (5), cyclo(L-Leu-L-Pro) cyclodipeptide (6), cyclo(L-Leu-L-Pro) diketopiperazine (7), 2,4-dihydroxypyrimidine (8), and 1H-indole-3-carbaldehyde (9). Compounds 1, 3, and 6 showed significant inhibition of papain, with IC50 values of 25.3 ± 1.9, 39.4 ± 2.5, and 7.4 ± 0.5 µM, respectively. Compound 1 also showed significant inhibition against human cathepsins V and B with IC50 of 46.0 ± 3.0 and 6.8 ± 0.7 µM, respectively. The inhibition of papain by mycotoxins (fusaric acid and beauvericin) may indicate a mechanism of Fusarium in the roles of infection process.

Acetylation of cedrelone increases its cytotoxic activity and reverts the malignant phenotype of breast cancer cells in 3D culture.

Cedrelone is a limonoid isolated from the plant Trichilia catigua (Meliaceae). Previous studies have demonstrated that cedrelone (1) has several damaging effects on triple negative breast tumor (TNBC) cell line MDA-MB-231. In this work we investigated two new derivatives of cedrelone, the acetate (1a) and the mesylate (1b), to examine whether their effects are improved in comparison to the lead molecule. Cedrelone acetate (1a) was the most cytotoxic compound on TNBC cells and was chosen for additional analyses in traditional two-dimensional (2D) monolayer cultures and three-dimensional (3D) assays. In 2D, 1a induced cell cycle arrest, apoptosis and inhibited essential steps of the metastasis process of the MDA-MB-231 cells, in vitro. Moreover, 1a was able to revert the malignant phenotype of the T4-2 cells in 3D. These effects were concomitant with the downregulation of EGFR, ß1-integrin and phospho-Akt, which could have resulted in a decrease of NFκB levels and MMP9 activity. These results suggest that 1a could be used as an important model for the design of a new drug to be applied in cancer treatment and be further studied in vivo for its antitumor and antimetastatic effects.

A new approach for identifying antagonism among fungi species and antifungal activity.

New antifungals are increasingly needed due to the emergence of resistant fungal strains. Traditional antifungal assays are laborious and require significant amounts of samples. The present work presents a new proposal to evaluate antifungal activity and antagonism among fungal species, based on experiments of fungal culture and co-culture, 1H NMR profile of fungal culture extracts and chemometrics. In order to develop the work, six axenic cultures of fungi that infested fruits (Fusarium guttiforme, Pestalotiopsis diospyri, Phoma caricae-papayae, Colletotrichum horii, Phytophthora palmivora, and C. gloeosporioides), and co-cultures of all possible combination among them were performed (totalizing 63 experiments). All fungal extracts were evaluated by 1H NMR followed by Principal Component Analyses (PCA) in order to determine spectral dissimilarity among the extracts. Results showed that 1H NMR data evaluated by PCA were capable to predict both antagonism and antifungal activity. Traditional antifungal in vitro assays of active and inactive extracts were also performed in order to prove the prediction made by PCA. The obtained data showed that the approach is an outstanding tool to simultaneously obtain and evaluate bioactive compounds because: it was able to predict the activity of five different extracts in a collection of sixty-three, which would be much more difficult and time consuming if applied randomly; most important antifungal extracts are indicated by PCA; hundreds of traditional in vitro assays are avoid; and, the method is very time and money saving.

Picraviane A and B: Nortriterpenes with limonoid-like skeletons containing a heptanolide E-ring system from Picramnia glazioviana.

Two highly oxygenated nortriterpenes, picraviane A and B, were isolated from the ethanolic extract of Picramnia glazioviana Engl. The structures were determined by analysis of HRMS and 2D NMR spectroscopic data. Single-crystal X-ray diffraction data was also obtained for picraviane B. The absolute configuration of both compounds were assigned by comparison of experimental and calculated vibrational and electronic circular dichroism spectroscopy, respectively. Picraviane A showed a moderate cytotoxic activity against the triple negative MDA-MB-231 breast cancer cell line. These compounds represent an undescribed class of natural products with limonoid-like skeletons containing a heptanolide as the E-ring.

Cinnamic acids derived compounds with antileishmanial activity target Leishmania amazonensis arginase.

This study describes the activity of five natural hydroxycinnamic acids and derived compound: caffeic (1), rosmarinic (2), chlorogenic (3), and cryptochlorogenic (4), acids and isoverbascoside (5). All compounds inhibited Leishmania amazonensis arginase with IC50 -in range of 1.5-11 µM. Compounds 2 and 5 also showed activity against promastigotes of L. amazonensis with IC50  = 61 (28-133) µM and IC50  = 14 (9-24) µM, respectively. Further computational studies applying molecular docking simulations were performed on the competitive inhibitors to gain insight into the molecular basis for arginase inhibition and could be exploited to the development of new antileishmanials drug targeting parasite arginase.

Isolation of Chavibetol and Methyleugenol from Essential Oil of Pimenta pseudocaryophyllus by High Performance Liquid Chromatography.

A high performance liquid chromatography (HPLC) method was developed for the simultaneous isolation, on a semi-preparative scale, of chavibetol and methyleugenol from the crude essential oil of P. pseudocaryophyllus leaves. The purity of the isolated compounds and their quantifications were developed using GC/FID. Chavibetol was isolated with high purity (98.7%) and mass recovery (94.6%). The mass recovery (86.4%) and purity (85.3%) of methyleugenol were lower than those of chavibetol. Both compounds were identified on the basis of spectral analysis. The results suggest that the method can provide chavibetol with high purity, mass recovery, and productivity from crude essential, which will be used in bioassays against stored insect pests.

Cathepsin D immobilized capillary reactors for on-flow screening assays.

The treatment of diseases using enzymes as targets has called for the development of new and reliable methods for screening. The protease cathepsin D is one such target involved in several diseases such as tumors, degenerative processes, and vital processes of parasites causing schistosomiasis. Herein, we describe the preparation of a fused silica capillary, cathepsin D (CatD)-immobilized enzyme reactor (IMER) using in a multidimensional High Performance Liquid Chromatography-based method (2D-HPLC) and zonal affinity chromatography as an alternative in the search for new ligands. The activity and kinetic parameters of CatD-IMER were evaluated by monitoring the product MOCAc-Gly-Lys-Pro-Ile-Leu-Phe (P-MOCAc) (KM = 81.9 ±â€¯7.49 µmol/L) generated by cleavage of the fluorogenic substrate MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(DNP)-d-Arg-NH2 (S-MOCAc). Stability studies have indicated that CatD-IMER retained 20% of activity after 5 months, a relevant result, because proteases are susceptible to autoproteolysis in solution assays with free enzyme. In the search for inhibitors, 12 crude natural product extracts were analyzed using CatD-IMER as the target, resulting in the isolation of different classes of natural products. In addition, 26 compounds obtained from different species of plants were also screened, demonstrating the efficiency and reproducibility of the herein reported assay even in the case of complex matrices such as plant crude extracts.

[10]-gingerol induces apoptosis and inhibits metastatic dissemination of triple negative breast cancer in vivo.

There is increasing interest in the use of non-toxic natural products for the treatment of various pathologies, including cancer. In particular, biologically active constituents of the ginger oleoresin (Zingiber officinale Roscoe) have been shown to mediate anti-tumour activity and to contribute to the anti-inflammatory, antioxidant, antimicrobial, and antiemetic properties of ginger. Here we report on the inhibitory properties of [10]-gingerol against metastatic triple negative breast cancer (TNBC) in vitro and in vivo. We show that [10]-gingerol concentration-dependently induces apoptotic death in mouse and human TNBC cell lines in vitro. In addition, [10]-gingerol is well tolerated in vivo, induces a marked increase in caspase-3 activation and inhibits orthotopic tumour growth in a syngeneic mouse model of spontaneous breast cancer metastasis. Importantly, using both spontaneous and experimental metastasis assays, we show for the first time that [10]-gingerol significantly inhibits metastasis to multiple organs including lung, bone and brain. Remarkably, inhibition of brain metastasis was observed even when treatment was initiated after surgical removal of the primary tumour. Taken together, these results indicate that [10]-gingerol may be a safe and useful complementary therapy for the treatment of metastatic breast cancer and warrant further investigation of its efficacy, either alone or in combination with standard systemic therapies, in pre-clinical models of metastatic breast cancer and in patients.