Dereplication of 4-Quinolone Alkaloids from Waltheria indica (Malvaceae) Tissues Using Molecular Network Tools.

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.

Secondary Metabolites Diversity of Aspergillus unguis and Their Bioactivities: A Potential Target to Be Explored.

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.

Identification and characterization of piperine synthase from black pepper, Piper nigrum L.

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.

Chemophenetic Significance of Anomalocalyx uleanus Metabolites are Revealed by Dereplication Using Molecular Networking Tools.

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.

Piper nigrum CYP719A37 Catalyzes the Decisive Methylenedioxy Bridge Formation in Piperine Biosynthesis.

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.

A piperic acid CoA ligase produces a putative precursor of piperine, the pungent principle from black pepper fruits.

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.

Trypanosoma cruzi: analysis of two different strains after piplartine treatment.

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.

Trypanosoma cruzi: analysis of two different strains after piplartine treatment

ABSTRACT 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.

In vitro efficacy of plant extracts and synthesized substances on Rhipicephalus (Boophilus) Microplus (Acari: Ixodidae).

Herbal drugs have been widely evaluated as an alternative method of parasite control, aiming to slow development of resistance and obtain low-cost biodegradable parasiticides. This study evaluated the in vitro efficacy on Rhipicephalus (Boophilus) microplus of extracts from Carapa guianensis seed oil, Cymbopogon martinii and Cymbopogon schoenanthus leaf essential oil, and Piper tuberculatum leaf crude extract and similar synthesized substances. In the immersion test, engorged females were evaluated in five dilutions ranging from 10% to 0.030625% concentration. In the larval test on impregnated filter paper, the concentration ranged from 10% to 0.02%. The treatments and controls were done in three replicates. Chemical analysis of the oils was performed by gas chromatography. The main compounds were oleic acid (46.8%) for C. guianensis and geraniol for C. martinii (81.4%), and C. schoenanthus (62.5%). The isolated and synthesized substances showed no significant effect on larvae and adult. C. martinii and P. tuberculatum showed the best efficacy on the engorged females. The LC(50) and LC(90) were 2.93% and 6.66% and 3.76% and 25.03%, respectively. In the larval test, the LC(50) and LC(90) obtained for C. martinii, P. tuberculatum, and C. schoenanthus were 0.47% and 0.63%, 0.41% and 0.79%, 0.57% and 0.96%, respectively. The fact that geraniol is present in greater quantities in C. martinii explains its higher activity in relation to C. shoenanthus. It is necessary to validate the in vivo use of safe and effective phytoparasiticidal substances. Efforts should be focused on developing formulations that enhance the efficacy in vivo and lengthen the residual period.

The anthelmintic effect of plant extracts on Haemonchus contortus and Strongyloides venezuelensis.

The indiscriminate use of anthelmintics has resulted in the establishment of parasite resistance. Thus, this study aimed to evaluate the in vitro antiparasitic effect of plant extracts on Haemonchus contortus in sheep and the in vivo effect on Strongyloides venezuelensis in Rattus norvegicus. The plant extracts from Piper tuberculatum, Lippia sidoides, Mentha piperita, Hura crepitans and Carapa guianensis, produced at different research institutions, were chemically analyzed and evaluated through the egg hatch test (EHT) and larval development test (LDT) in H. contortus. P. tuberculatum (150 and 250 mg kg(-1) of body weight) was evaluated for its anthelmintic action on R. norvegicus experimentally infected with S. venezuelensis. In the EHT, the LC(50) and LC(90) of the extracts were respectively as follows: 0.031 and 0.09 mg mL(-1) for P. tuberculatum, 0.04 and 0.13 mg mL(-1) for L. sidoides, 0.037 and 0.10 mg mL(-1) for M. piperita, 2.16 and 17.13 mg mL(-1) for H. crepitans and 2.03 × 10(-6) and 1.22 × 10(-12) mg mL(-1) for C. guianensis. In the LDT, the LC(50) and LC(90) were respectively: 0.02 and 0.031 mg mL(-1) for P. tuberculatum, 0.002 and 0.04 mg mL(-1) for L. sidoides, 0.018 and 0.03 mg mL(-1) for M. piperita, 0.36 and 0.91 mg mL(-1) for H. crepitans and 17.65 and 1890 mg mL(-1) for C. guianensis. The extract of P. tuberculatum showed the following substances: piperamides as (Z)-piplartine, (E)-piplartine, 8,9-dihydropiplartine, piperine, 10,11-dihydropiperine, 5,6 dihydropiperlongumine and pellitorine. The major compounds of the oils were thymol (76.6%) for L. sidoides, menthol (27.5%) for M. piperita and oleic acid (46.8%) for C. guianensis. Regarding the in vivo test, neither dose of P. tuberculatum caused any significant reduction (P>0.05) in worm burden and fecal egg counts compared with the control group. We conclude that the extracts of P. tuberculatum, L. sidoides and M. piperita have effective activity when tested in vitro, but the doses of the extract of P. tuberculatum have no effect when employed in in vivo tests.

Trypanocidal activity of Piper arboreum and Piper tuberculatum (Piperaceae) / Atividade tripanocida de Piper arboreum e Piper tuberculatum (Piperaceae)

No escopo de nossas pesquisas sobre agentes bioativos da flora brasileira, vinte e quatro extratos e frações de Piper arboreum Aub. e Piper tuberculatum Jacq. (Piperaceae) tiveram sua atividade tripanocida avaliada através do ensaio colorimétrico com MTT. As atividades mais potentes foram manifestadas pelas frações hexânicas das folhas de P. arboreum (CI50 = 13,3 µg/mL) e P. tuberculatum (CI50 = 17,2 µg/mL). As frações hexânicas dos frutos de P. tuberculatum e P. arboreum também apresentaram efeito tóxico potente contra as formas epimastigotas de Trypanosoma cruzi, com valores de CI50 (µg/mL) de 32,2 e 31,3, respectivamente. Adicionalmente, o estudo fitoquímico da fração hexânica das folhas de P. arboreum forneceu duas amidas pirrolidínicas, piperilina (1) e 4,5-diidropiperilina (2), que podem ser responsáveis pela atividade antiprotozoária desta fração.

In the scope of our ongoing research on bioactive agents from Brazilian flora, twenty-four extracts and fractions obtained from Piper arboreum Aub. and Piper tuberculatum Jacq. (Piperaceae) were screened for trypanocidal activity by using MTT colorimetric assay. The strongest activity was found in hexane fractions from the leaves of P. arboreum (IC50= 13.3 µg/ mL) and P. tuberculatum (IC50 = 17.2 µg/mL). Hexane fractions of the fruits of P. tuberculatum and P. arboreum showed potent toxic effects on epimastigote forms of Trypanosoma cruzi, with values of IC50 (µg/mL) of 32.2 and 31.3, respectively. Additionally, the phytochemical study of the hexane fraction of P. arboreum leaves furnished two pyrrolidine amides, piperyline (1) and 4,5-dihydropiperyline (2), which could be responsible, at least in part for the observed antiprotozoal activity.