Waltheria indica L. (Malvaceae) is a medicinal plant widely used in traditional medicine and recognized as a rich source of bioactive alkaloids. Despite extensive phytochemical investigations, information regarding the spatial distribution of these specialized metabolites within plant tissues remains limited. This study aimed to investigate the tissue-specific localization of alkaloids in W. indica. using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). A preliminary study was conducted during method development to establish suitable sample preparation and matrix application conditions using leaves, stems, and flowers collected in Guadalupe (Rio de Janeiro, Brazil). The conditions selected from the exploratory assays were subsequently applied to cryosectioned leaves, stems, and roots, followed by MALDI FT-ICR MSI analysis in positive ion mode. MALDI-MSI revealed a heterogeneous and tissue-dependent distribution of alkaloids across different plant organs. Roots exhibited a broader diversity of alkaloid-related ions, while stems showed preferential localization in vascular tissues. In leaves, specific alkaloids were predominantly localized in the midrib and mesophyll regions. These spatial patterns were consistent across samples and correlated with the anatomical organization of each organ. Overall, this study provides the first spatially resolved chemical maps of alkaloids in W. indica, demonstrating that metabolite accumulation is strongly associated with tissue type. These findings highlight the potential of MALDI-MSI as a powerful tool to link chemical diversity with plant anatomy, offering new insights into the functional organization of alkaloid biosynthesis and accumulation in medicinal plants.

Lepidopteran pests cause severe global economic damage; they are currently mitigated by synthetic pesticides that trigger widespread resistance and environmental toxicity. This systematic review evaluates the potential of Piper essential oils (EOs) as high-performance, sustainable bio-based insecticides, aligning with the 12 Principles of Green Chemistry. Analyzing studies covering Piper species, we identified phenylpropanoids (e.g., dillapiole and safrole) and terpenoids as key biodegradable scaffolds for pest management. The results highlight P. aduncum and P. divaricatum for their exceptional efficacy against Spodoptera frugiperda and Plutella xylostella, often exhibiting toxicity levels comparable to botanical standards like azadirachtin. Crucially, this review reveals that Piper EOs can outperform the synthetic industrial synergist piperonyl butoxide (BPO), with natural binary mixtures enhancing insecticidal potency by up to 11-fold. Furthermore, specific EOs contribute to a preventative green strategy by causing the structural disintegration of the egg chorion. By focusing on renewable biomass and design for degradation (Principles 7 and 10), this work anchors the Piper genus as a cornerstone for the circular bioeconomy and sustainable agricultural innovation, reducing the chemical footprint of modern crop protection.

Waltheria indica (Malvaceae) is a plant popularly used in folk medicine by traditional African and indigenous communities, and in various countries worldwide, to treat general inflammation. Several biological activities of this plant have been reported, including acetylcholinesterase inhibition and potential anti-human immunodeficiency virus (HIV), antinociceptive, analgesic, antifungal, anticancer, anti-inflammatory, leishmanicidal, trypanocidal, antioxidant, and antibacterial activities. The chemical profile of Waltheria indica was assessed by dereplication analysis using UPLC-MS/MS, and data acquisition was performed using chemoinformatics tools, such as Mass Spectrometry-Data Independent AnaLysis (MS-DIAL) and MS-FINDER softwares. The preprocessed data were sent to the GNPS to build a feature-based molecular network (FBMN). Thirty-three 4-quinolone alkaloids were annotated in the extracts and fractions of stems and roots, whereas 12 were annotated in the extracts and fractions of flowers and leaves. This represents an inaugural chemical investigation study employing UPLC-Q-TOF-MS/MS analysis, along with a molecular network approach, within this species and genus.

Waltheria, a genus within the Malvaceae family, is abundantly distributed in tropical and subtropical areas worldwide. Many species of this genus are widely utilized in various ways, including chewing, in folk medicine, acting as an anti-inflammatory agent, and treating gastrointestinal disorders, rheumatism, and asthma, among other conditions. These applications are largely due to their secondary metabolites, primarily quinolone alkaloids and cyclopeptides. Several biological activities have been reported for Waltheria species, including antifungal, anticancer, trypanocidal, acetylcholinesterase inhibitory, potential anti-HIV, antinociceptive, analgesic, anti-inflammatory, antibacterial, antioxidant, and leishmanicidal activities. This review not only presents information on isolated alkaloids and their biological activities but also delves into biosynthetic, chemosystematic, medicinal chemistry, and total synthesis aspects. Additionally, the manuscript highlights other applications of alkaloids of the genus, such as a study on their herbicidal activity, which shows significant potential for agricultural use.

Several Piper species accumulate piperamides as secondary metabolites, and although they have relevant biological importance, many details of their biosynthetic pathways have not yet been described experimentally. Experiments involving enzymatic reactions and labeled precursor feeding were performed using the species Piper tuberculatum and Piper arboreum. The activities of the phenylalanine ammonia lyase (PAL) enzymes, which are involved in the general phenylpropanoid pathway, were monitored by the conversion of the amino acid L-phenylalanine to cinnamic acid. The activity of the 4-hydroxylase (C4H) enzyme was also observed in P. tuberculatum by converting cinnamic acid to p-coumaric acid. L-[UL-14C]-phenylalanine was fed into the leaves of P. tuberculatum and incorporated into piperine (1), 4,5-dihydropiperine (2), fagaramide (4), trans-piplartine (7), and dihydropiplartine (9). In P. arboreum, it was only incorporated into the piperamide 4,5-dihydropiperiline (3). L-[UL-14C]-lysine was successfully incorporated into the 4,5-dihydropiperine piperidine group (2), dihydropyridinone, and trans- (7) and cis-piplartine (8). These data corroborate the proposal of mixed biosynthetic origin of piperamides with the aromatic moiety originating from cinnamic acid (shikimic acid pathway) and key amide construction with amino acids as precursors.

Cancer therapy represents a challenge, even with the current scientific developments. Over the years, several studies have shown that the application of antitumor drug nanocarriers is an efficient strategy; however, it requires further improvement. Several nanosystems have been developed, some of which are already in use, including liposomes. Polymer-based nanocarriers are still being adjusted to the peculiarities of the human body due to the biological barriers that were encountered by the first systems that were developed. Among them, the formation of corona proteins, clearance by the endothelial reticulum system and kidneys, activation of the immune system, lack of selectivity, and difficult release have been extensively studied and improved with the development of new devices. In this review, we explore the evolution of primary nanocarriers based on polymeric micelles and highlight the gaps that remain in this field to assist in the research of new systems with superior therapeutic indices.

Aspergillus unguis belongs to the Aspergillus section Nidulantes. This species is found in soils and organisms from marine environments, such as jellyfishes and sponges. The first chemical study reported in the literature dates from 1970, with depsidones nidulin (1), nornidulin (2), and unguinol (3) being the first isolated compounds. Fifty-two years since this first study, the isolation and characterization of ninety-seven (97) compounds have been reported. These compounds are from different classes, such as depsides, depsidones, phthalides, cyclopeptides, indanones, diarylethers, pyrones, benzoic acid derivatives, orcinol/orsenillate derivatives, and sesterpenoids. In terms of biological activities, the first studies on isolated compounds from A. unguis came only in the 1990s. Considering the tendency for antiparasitic and antibiotics to become ineffective against resistant microorganisms and larvae, A. unguis compounds have also been extensively investigated and some compounds are considered very promising. In addition to these larvicidal and antimicrobial activities, these compounds also show activity against cancer cell lines, animal growth promotion, antimalarial and antioxidant activities. Despite the diversity of these compounds and reported biological activities, A. unguis remains an interesting target for studies on metabolic induction to produce new compounds, the determination of new biological activities, medicinal chemistry, structural modification, biotechnological approaches, and molecular modeling, which have yet to be extensively explored.

A chemical investigation of the stem of Waltheria indica (Malvaceae) yielded twelve 4-quinolone alkaloids, which were primarily waltheriones. These were waltherione A (1), waltherione B (2), waltherione C (3), waltherione G (4), waltherione H (5), waltherione J (6), waltherione L (7), waltherione P (8), chamaedrone (9), 8-deoxy-antidesmone (10), antidesmone (11), and the previously unreported alkaloid N-methoxy-waltherione A (12). These alkaloids belong to an unusual class of 4-quinolones and therefore, have chemosystematic significance for distinguishing the Waltheria and Melochia genera from the rest of the Malvaceae family. The ability of the alkaloid isolates to reverse the phenotypic expression of fluconazole-resistance was tested by using a mutant strain of Saccharomyces cerevisiae that expressed a Candida albicans transporter. Of the isolates tested, waltherione G afforded a positive result. Leishmanicidal activity and bactericidal tests were also performed using the isolated alkaloids, which showed promising results.

Anomalocalyx uleanus (Pax & K. Hoffm.) Ducke (Euphorbiaceae) is a singular species in the genus and is restricted and exclusive to the Brazilian Amazon. A phytochemical study of A. uleanus leaves was performed, yielding the isolation of five major compounds: catechin/epicatechin, afzelin, quercetin 3-O-α-L-rhamnopyranoside, and astilbin. The phytochemical compositions of the methanolic extracts of leaves, roots, bark, and stem bark were determined using a dereplication approach. Forty-six compounds were annotated from the liquid chromatography-mass spectrometry (LC-MS/MS) data, while four lipids were identified using gas chromatography-mass spectrometry (GC-MS). In total, fifty compounds were detected, and they belonged to the primary metabolism and several classes of natural products such as flavonoids, flavonoids O-glycosides, flavonoids C-glycosides, biflavonoids, procyanidin, triterpene, triterpenes esterified with phenylpropanoids, phenylpropanoid derivatives, flavonolignans, coumarins, quinic acid derivatives, and benzoic acid derivatives. This is the first report on the phytochemical data of the genus Anomalocalyx, and the results of this study will contribute to the chemosystematic knowledge of the Euphorbiaceae family and justify the need for investigation of the pharmacological potential of the species A. uleanus.

Black pepper ( Piper nigrum L.) is the world’s most popular spice and is also used as an ingredient in traditional medicine. Its pungent perception is due to the interaction of its major compound, piperine (1-piperoyl-piperidine) with the human TRPV-1 or vanilloid receptor. We now identify the hitherto concealed enzymatic formation of piperine from piperoyl coenzyme A and piperidine based on a differential RNA-Seq approach from developing black pepper fruits. This enzyme is described as piperine synthase (piperoyl-CoA:piperidine piperoyl transferase) and is a member of the BAHD-type of acyltransferases encoded by a gene that is preferentially expressed in immature fruits. A second BAHD-type enzyme, also highly expressed in immature black pepper fruits, has a rather promiscuous substrate specificity, combining diverse CoA-esters with aliphatic and aromatic amines with similar efficiencies, and was termed piperamide synthase. Recombinant piperine and piperamide synthases are members of a small gene family in black pepper. They can be used to facilitate the microbial production of a broad range of medicinally relevant aliphatic and aromatic piperamides based on a wide array of CoA-donors and amine-derived acceptors, offering widespread applications.

Black pepper (Piper nigrum) is among the world's most popular spices. Its pungent principle, piperine, has already been identified 200 years ago, yet the biosynthesis of piperine in black pepper remains largely enigmatic. In this report we analyzed the characteristic methylenedioxy bridge formation of the aromatic part of piperine by a combination of RNA-sequencing, functional expression in yeast, and LC-MS based analysis of substrate and product profiles. We identified a single cytochrome P450 transcript, specifically expressed in black pepper immature fruits. The corresponding gene was functionally expressed in yeast (Saccharomyces cerevisiae) and characterized for substrate specificity with a series of putative aromatic precursors with an aromatic vanilloid structure. Methylenedioxy bridge formation was only detected when feruperic acid (5-(4-hydroxy-3-methoxyphenyl)-2,4-pentadienoic acid) was used as a substrate, and the corresponding product was identified as piperic acid. Two alternative precursors, ferulic acid and feruperine, were not accepted. Our data provide experimental evidence that formation of the piperine methylenedioxy bridge takes place in young black pepper fruits after a currently hypothetical chain elongation of ferulic acid and before the formation of the amide bond. The partially characterized enzyme was classified as CYP719A37 and is discussed in terms of specificity, storage, and phylogenetic origin of CYP719 catalyzed reactions in magnoliids and eudicots.

Black pepper (Piper nigrum L.) is known for its high content of piperine, a cinnamoyl amide derivative regarded as largely responsible for the pungent taste of this widely used spice. Despite its long history and worldwide use, the biosynthesis of piperine and related amides has been enigmatic up to now. In this report we describe a specific piperic acid CoA ligase from immature green fruits of P. nigrum. The corresponding enzyme was cloned and functionally expressed in E. coli. The recombinant enzyme displays a high specificity for piperic acid and does not accept the structurally related feruperic acid characterized by a similar C-2 extension of the general C6-C3 phenylpropanoid structure. The enzyme is also inactive with the standard set of hydroxycinnamic acids tested including caffeic acid, 4-coumaric acid, ferulic acid, and sinapic acid. Substrate specificity is corroborated by in silico modelling that suggests a perfect fit for the substrate piperic acid to the active site of the piperic acid CoA ligase. The CoA ligase gene shows its highest expression levels in immature green fruits, is also expressed in leaves and flowers, but not in roots. Virus-induced gene silencing provided some preliminary indications that the production of piperoyl-CoA is required for the biosynthesis of piperine in black pepper fruits.

Piper tuberculatum (Piperaceae) is a species that accumulates especially amides as secondary metabolites and several biological activities was previously reported. In this article, we report a proteomic study of P. tuberculatum. Bidimensional electrophoresis (2D SDS-PAGE) and mass spectrometry (ESI-Q-TOF) were used in this study. Over a hundred spots and various peptides were identified in this species and the putative functions of these peptides related to defense mechanism as biotic and abiotic stress were assigned. The information presented extend the range of molecular information of P. tuberculatum.

The hemoflagellate protozoan, Trypanosoma cruzi, mainly transmitted by triatomine insects through blood transfusion or from mother-to-child, causes Chagas' disease. This is a serious parasitic disease that occurs in Latin America, with considerable social and economic impact. Nifurtimox and benznidazole, drugs indicated for treating infected persons, are effective in the acute phase, but poorly effective during the chronic phase. Therefore, it is extremely urgent to find innovative chemotherapeutic agents and/or effective vaccines. Since piplartine has several biological activities, including trypanocidal activity, the present study aimed to evaluate it on two T. cruzi strains proteome. Considerable changes in the expression of some important enzymes involved in parasite protection against oxidative stress, such as tryparedoxin peroxidase (TXNPx) and methionine sulfoxide reductase (MSR) was observed in both strains. These findings suggest that blocking the expression of the two enzymes could be potential targets for therapeutic studies.

The known kavalactones (E)-4-methoxy-6-styryl-2H-pyran-2-one, 4-methoxy-6-(3-phenyloxiran-2-yl)-2H-pyran-2-one, 6-(1,2-dihydroxy-2-phenylethyl)-4-methoxy-2H-pyran-2-one, the three benzoic acid derivatives methyl-4-methoxy-3-(3'-methyl-2'-butenyl)benzoate and methyl 2,2-dimethyl-4-oxochroman-6-carboxylate, and a new methyl 4-methoxy-3-(3-methylbut-2-enoyl)benzoate were isolated from the ethanolic extract of Piper fuligineum.The structures of these compounds were determined by using a combination of spectroscopic methods, including 1D-and 2D-nuclear magnetic resonance spectroscopy and high-resolution mass spectrometry.This is the first report of the chemical study of P. fuligineum, and the methyl 4-methoxy-3-(3-methylbut-2-enoyl)benzoate is described as a new natural product.

Piperlongumine is a natural amide alkaloid isolated from several species of Piper and is described in the literature as selectively cytotoxic to several cancer cell lines.Inhibiting cell migration has gained considerable interest as an approach for discovering antimetastatic agents because this process is fundamental to metastasis.Piperlongumine, selected from cell-based assay screening of NuBBE Database, inhibited the migration of MDA-MB-231 breast cancer cells with an EC 50 of 3.0 ± 1.0 µM by the Boyden chamber assay.A series of five analogous compounds based on the structure of piperlongumine were designed, synthesized and evaluated in cell migration and cytotoxicity assays.The analogue designed by molecular simplification ((E)-N-acryloyl-3-(3,4,5-trimethoxyphenyl)acrylamide) was the most active of the series, with an EC 50 of 1.5 ± 1 µM.Additionally, this compound was selectively cytotoxic, with a selectivity index (SI) of 4.4.

The first step to consider in a proteomic study is the harvesting and preparation of the samples. Meticulous care is essential in the preparation of high-purity samples in order to achieve quality results. The next step involves the protein separation processes: the protein fraction may be subjected to one-dimensional electrophoresis in polyacrylamide gel (SDS-PAGE) – which separates the proteins by molecular mass – or to two-dimensional electrophoresis in polyacrylamide gel (2D-PAGE) – which separates them by molecular mass and isoelectric point. The gel areas containing the protein may be excised and subjected to a trypsinization procedure, which will yield a peptide mixture. The peptides derived from a given spot or band can then be analyzed by mass spectrometry (MS); a preceding separation may be carried out using liquid chromatography. The chapter describes the primary characteristics of the analytical separation, detection, and identification tools used in proteome- and metabolome-related studies.

Sugarcane is an important commercial crop cultivated in tropical and sub-tropical regions mainly for its sucrose content, the raw material for sugar and ethanol industries. Brazil is the largest producer of ethanol from sugarcane in the world and occupies the leadership in technology for its production. In Brazil, sugarcane cultivation is expanding to central and northeast regions, where water availability is scarce. Sugarcane production is negatively influenced by a vast number of environmental factors that affect growth, metabolism and yield. Among them, drought is the strongest and has the most severe limitation on sugarcane yield [1]. Water stress induces various biochemical and physiological responses in plants, such as accumulation of solutes including sugars, polyols, betaines and amino acids. The capacity of monitoring a set of metabolites could largely improve the understanding of mechanisms involved in plant responses to drought stress. Besides, differences in metabolite content can also represent good predictors for drought tolerant phenotypes both for variety screening and plant breeding programs. Here we report the analysis of leaf sugarcane (+1), from a five month-old drought tolerant variety (CTC 15), submitted to normal water supply, moderate stress (40% field capacity) and severe stress (20% field capacity). Leaf metabolites were extracted from 50 mg of powder tissue, according to De Vos et al. [2] with minor modifications. All treatments were performed in nine biological replicates. Samples were analyzed by UPLC-Q-TOF-MS and the range of the mass scan was 100-2000 m/z, in positive mode. Reverse-phased chromatography was performed using the following gradient condition: 95% A (H2O+0,1% HCOOH) and 5% B (ACN+0,1% HCOOH) for 6 minutes, 25% A and 75% B for 6 minutes, 5% A and 95% B for 1 minute. Data processing and multivariate analysis were performed in MarkerLynx and MetaboAnalyst softwares. We were able to discriminate samples and observe leaf metabolite changes in response to water stress. PLSDA model demonstrated a clear separation between samples (Q2>0.9). Besides, metabolites were ranked according to their contribution to the prediction of drought tolerance, by the “variable importance in the projection” (VIP). These results provide important insights into sugarcane responses to water stress. The next steps aim to identify differentially abundant metabolites. Financial Support: FAPESP/Process number: 2012/22227-4

Sugarcane (Saccharum spp.) is one of the most important cultivated grasses of the world and Brazil is the largest producer, with the Sao Paulo state concentrating more than half area for this crop. The genetic mechanisms that control sugarcane sucrose production have been studied at various levels, such as gene identification and localization, identification of quantitative trait locus controlling, transcriptome and proteome. Thus, an understanding of the mechanisms that regulate the sucrose production and accumulation is an interesting approach to target higher sugar yield in this plant. This work aims to identify the metabolite fingerprint during plant development and to correlate it with sucrose production and accumulation. In order to elucidate this mechanism in the metabolic level, we used the leaf +1 of the sugarcane variety SP80-3280 grown in the field at 3, 7 and 11 months after planting. Metabolite extraction was made from 50 mg of tissue using an extraction solution composed of 99.875% (v/v) of methanol and 0.125% (v/v) of formic acid, following the protocol used by De Vos et al. [1] with some modifications. The samples were analyzed by UPLC-ESI-QTOF-MS (Waters Technologies, UK), in positive mode in a total of six biological replicates and three technical replicates for each sampling period. Data processing was performed using the software XCMS [2] and multivariate statistical analysis by Principal Component Analysis (PCA). The sum of the two main components of the PCA (55.48%) ensured a reliable analysis. PCA highlighted groups of samples related to the sampling time, mainly at 3 and 11 month-old. Visualizing the differences between 3 and 11 months an OPLS-DA was performed and we observed the main markers responsible for this differentiation. In a further analysis these markers will be identified and potentially correlated with sucrose production and accumulation in sugarcane.