Inhibitory effects against SARSCoV-2 main protease (Mpro) of biflavonoids and benzophenones from the fruit of Platonia insignis.

The SARS-CoV-2 mutation and the limitation of the approved drug against COVID-19 are still a challenge in many country healthcare systems and need to be affronted despite the set of vaccines to prevent this viral infection. To contribute to the identification of new antiviral agents, the present study focused on natural products from an edible fruit with potential inhibitory effects against the SARS-CoV-2 main protease (Mpro). First, LC-ESIMS analysis of Platonia insignis fruits was performed and showed the presence of biflavonoids and benzophenones in the seed and pulp, respectively. Then, maceration and chromatographic purification led to the identification of two triglycerides (1 and 2) alongside chamaejasmine (3) and volkensiflavone (4) from the seed and isogarcinol (5) and cycloxanthochymol (6), from the pulp. Compounds 1-6 after evaluating their inhibitory against Mpro, displayed from no to significant activity. Compound 5 was the most potent with an IC50 value of 0.72 µM and was more active than the positive control, Ebselen (IC50 of 3.4 µM). It displayed weak and no cytotoxicity against THP-1 (CC50 of 116.2 µM) and Vero cell lines, respectively. Other active compounds showed no cytotoxicity against THP-1. and Vero cell lines. Molecular docking studies revealed interactions in the catalytic pocket between compound 5 and amino acid residues that composed the catalytic dyads (His 41 and Cyst 145).

Anti-Candida albicans Activity of Ononin and Other Secondary Metabolites from Platonia Insignis MART.

Candida albicans is a human pathogen that is part of the healthy microbiome. However, it is often associated with opportunistic fungal infections. The treatment of these infections is challenging because prolonged exposure to antifungal drugs can culminate in fungal resistance during therapy, and there is a limited number of available drugs. Therefore, this study investigated the antifungal activity of ononin by in silico and in vitro assays, and in Tenebrio molitor as an alternative in vivo model of infection caused by C. albicans. Ononin is an isoflavone glycoside derived from formononetin that has various biological activities. According in silico evaluation, ononin showed the best electron affinity in molecular docking with CaCYP51, with a binding free energy of -10.89 kcal/mol, superior to that of the antifungal drugs fluconazole and posaconazole. The ononin + CaCYP51 complex formed hydrogen bonds with Tyr132, Ser378, Phe380, and Met508, as well as hydrophobic connections with Tyr118, Leu121, Phe126, Leu131, Ile304, and Leu309, and interactions with the heme group. Ononin exerted anti-Candida albicans activity, with MIC between 3.9 and 7.8 µg/mL, and inhibited young and mature biofilms, with a reduction in cell density and metabolic activity of 50 to 80%. The compound was not cytotoxic to sheep red blood cells at concentrations up to 1000 µg/mL. Larvae of the mealworm T. molitor were used as an alternative in vivo model of C. albicans infection. Ononin was able to prolong larval survival at concentrations of 0.5, 1, and 5 mg/kg, and was not toxic up to a concentration of 20 mg/kg. Moreover, ononin reduced the fungal charge in treated animals. In conclusion, our results suggest that ononin has anti-Candida albicans activity and is a potential candidate for the development of new therapeutic alternatives.

Amazonian Bacuri (Platonia insignis Mart.) Fruit Waste Valorisation Using Response Surface Methodology.

Bacuri (Platonia insignis Mart) is a species from the Clusiaceae genus. Its fruit pulp is commonly used in South America in several food products, such as beverages, ice cream and candies. Only the pulp of the fruit is used, and the peels and seeds are considered waste from these industries. As a trioxygenated xanthone source, this species is of high interest for bioproduct development. This work evaluated the mesocarp and epicarp of bacuri fruits through different extraction methods and experimental conditions (pH, temperature and solvent) in order to determine the most effective method for converting this agro-industrial waste in a value-added bioproduct. Open-column procedures and HPLC and NMR experiments were performed to evaluate the chemical composition of the extracts, along with total phenols, total flavonoids and antioxidant activities (sequestration of the DPPH and ABTS radicals). A factorial design and response surface methodology were used. The best extraction conditions of substances with antioxidant properties were maceration at 50 °C with 100% ethanol as solvent for mesocarp extracts, and acidic sonication in 100% ethanol for epicarp extracts, with an excellent phenolic profile and antioxidant capacities. The main compounds isolated were the prenylated benzophenones garcinielliptone FC (epicarp) and 30-epi-cambogin (mesocarp). This is the first study analysing the performance of extraction methods within bacuri agro-industrial waste. Results demonstrated that shells and seeds of bacuri can be used as phenolic-rich bioproducts obtained by a simple extraction method, increasing the value chain of this fruit.

Biflavones from Platonia insignis Mart. Flowers Promote In Vitro Antileishmanial and Immunomodulatory Effects against Internalized Amastigote Forms of Leishmania amazonensis.

Leishmaniasis is an infectious disease that affects millions of people worldwide, making the search essential for more accessible treatments. The species Platonia insignis Mart. (Clusiaceae) has been extensively studied and has gained prominence for its pharmacological potential. The objective of this work was to evaluate the antileishmania activity, cytotoxic effect and activation patterns of macrophages of hydroalcoholic extract (EHPi), ethyl acetate fractions (FAcOEt) and morelloflavone/volkensiflavone mixture (MB) from P. insignis flowers. EHPi, FAcOEt and MB demonstrated concentration-dependent antileishmania activity, with inhibition of parasite growth in all analyzed concentrations. EHPi exhibited maximum effect at 800 µg/mL, while FAcOEt and MB reduced the growth of the parasite by 94.62% at 800 µg/mL. EHPi, FAcOEt and MB showed low cytotoxic effects for macrophages at 81.78, 159.67 and 134.28 µg/mL, respectively. EHPi (11.25 µg/mL), FAcOEt (11.25 and 22.5 µg/mL) and MB (22.5 µg/mL) characterized the increase in lysosomal activity, suggesting a possible modulating effect. These findings open for the application of flowers from a P. insignis flowers and biflavones mixture thereof in the promising treatment of leishmaniasis.

Modulation of the Drug Resistance by Platonia insignis Mart. Extract, Ethyl Acetate Fraction and Morelloflavone/Volkensiflavone (Biflavonoids) in Staphylococcus aureus Strains Overexpressing Efflux Pump Genes.

BACKGROUND: Microbial resistance to antibiotics is a global public health problem, which requires urgent attention. Platonia insignis is a native species from the eastern Brazilian Amazon, used in the treatment of burns and wounds. OBJECTIVES: To evaluate the antimicrobial activity of the hydroalcoholic extract of P. insignis (PIHA), the ethyl acetate fraction (PIAE), and its subfraction containing a mixture of biflavonoids (BF). Moreover, the effect of these natural products on the antibiotic activity against S. aureus strains overexpressing efflux pump genes was also evaluated. METHODS: Minimal inhibitory concentrations were determined against different species of microorganisms. To evaluate the modulatory effect on the Norfloxacin-resistance, the MIC of this antibiotic was determined in the absence and presence of the natural products at subinhibitory concentrations. Inhibition of the EtBr efflux assays were conducted in the absence or presence of natural products. RESULTS: PIHA showed a microbicidal effect against S. aureus and C. albicans, while PIAE was bacteriostatic for S. aureus. PIAE and BF at subinhibitory concentrations were able to reduce the MIC of Norfloxacin acting as modulating agents. BF was able to inhibit the efflux of EtBr efflux in S. aureus strains overexpressing specific efflux pump genes. CONCLUSION: P. inignisis, a source of efflux pump inhibitors, including volkensiflavone and morelloflavone, which were able to potentiate the Norfloxacin activity by NorA inhibition, being also able to inhibit QacA/B, TetK and MsrA. Volkensiflavone and morelloflavone could be used as an adjuvant in the antibiotic therapy of multidrug resistant S. aureus strains overexpressing efflux pumps.

Antifungal and Antivirulence Activities of Hydroalcoholic Extract and Fractions of Platonia insignis Leaves against Vaginal Isolates of Candida Species.

Vulvovaginal candidiasis is a common fungal infection in women. In this study, Platonia insignis hydroalcoholic extract (PiHE) and its fractions were evaluated for antifungal and antivirulence activities against vaginal Candida species. Dichloromethane (DCMF) and ethyl acetate fractions (EAF) obtained from PiHE effectively inhibited the pathogen. Electrospray ionization mass spectrometry was used for identifying the main compounds in extracts. Minimal inhibitory and fungicidal concentrations (MIC and MFC, respectively) were determined by a broth microdilution assay. Furthermore, we evaluated the effect of the extract and fractions on the virulence properties of Candida albicans, and their cytotoxicity effect was determined on RAW 264.7 cells. Compounds found in extracts were flavonoid glycosides, mainly derivatives of quercetin and myricetin. Extracts showed antifungal potential, with the lowest MIC value for EAF (1.3 mg/mL) and inhibited Candida adherence and biofilm formation. EAF disrupted 48 h biofilms with an inhibition rate of more than 90%. The extract and its fractions exhibited no cytotoxicity. The antifungal effects were attributed to the ability of these extracts to alter the mitochondrial membrane potential for the release of pro-apoptotic factors in the cytosol. In conclusion, our data suggest that PiHE and EAF could act as novel candidates for the development of new therapeutic treatments against fungal infections.

Immunomodulatory and toxicological evaluation of the fruit seeds from Platonia insignis, a native species from Brazilian Amazon Rainforest

Abstract The “bacuri” (Platonia insignis Mart., Clusiaceae) is a native tropical fruit from the Brazilian Amazon and Northeast Regions. Its seeds are used to treat inflammatory diseases, diarrhea and skin problems in traditional medical practices. Regarding its widespread medicinal uses, it is important to evaluate the biological and toxicological potential of this species. This way, the aim of this study was to investigate the in vitro cytotoxic and immunomodulatory effects of the hexanic extract of P. insignis seeds, as well as its in vivo acute oral toxicity. The biological evaluation was performed by the determination of cytotoxic (MTT and hemolysis assay) and immunomodulatory (phagocytic capacity, lysosomal volume and nitrite production) activities of EHSB in murine peritoneal macrophages. In addition, the oral acute toxicity was evaluated using female Wistar rats treated with EHSB (2.0 g/kg), in accordance with the OECD 423 Guideline. The EHSB showed low toxicity for macrophages in the MTT test (CC50 value: 90.03 µg/ml), as well as for erythrocytes, which caused only 2.5% hemolysis at the highest concentration. A strong immunomodulatory activity was observed by a markedly increase of the NO production, phagocytic ability and lysosomal volume. On the other hand, it was not observed deaths or changes in the clinical and behavioral parameters in the toxicological evaluation. This manner, the present study contributes to the knowledge about the immunomodulatory and toxicological properties of the P. insignis. This may provide perspectives for the evaluation and development of effective and safe phytomedicines created from the Brazilian local biodiversity.

Ensaios pré-clínicos em ratos tratados com 1, 3-diestearil-2-oleilglicerol, constituinte isolado de Platonia insignis / Pre clinical trials in rats treated with 1, 3 -distearoyl-2-oleoylglycerol (TG1) constituent isolated from Platonia insignis

Introdução: Avaliou-se a toxicidade aguda de 1,3-diestearil-2-oleil-glicerol (TG1), composto obtido de Platonia insignis Mart. (bacurizeiro), após administração oral em ratos Wistar. Métodos: A toxicidade aguda foi analisada através dos parâmetros hematológicos e bioquímicos. A análise de citotoxicidade in vitro foi feita pelo método do sal 3-(4,5-dimetil-2-tiazol)-2,5-difenil-2-H-brometo de tetrazolium (MTT). Os tecidos cerebrais e hepáticos foram avaliados histopatologicamente. Resultados: O tratamento agudo com TG1(dose de 30 mg kg -1) não produziu alterações hematológicas e histopatológicas nas áreas cerebrais e hepáticas. A redução dos níveis das enzimas transaminase (AST) e fosfatase alcalina (ALKP) pode sugerir proteção hepática. As análises bioquímicas da aspartato aminotransferase, ALKP e do ácido úrico apresentaram seus níveis reduzidos, conferindo preservação dos rins e fígado dos animais (p<0,05). TG1 não revelou potencial citotóxico pelo método MTT. Conclusão: O tratamento com TG1 não produz alterações hematológicas, bioquímicas, histopatológicas cerebrais e hepáticas em ratos o que caracteriza uma baixa toxicidade.

Introduction: The aim of this study was to assess the acute toxicity of 1,3-distearoyl-2-oleoylglycerol (TG1), a compound isolated from Platonia insignis Mart. (bacurizeiro). Methods: The acute toxicity was analyzed by biochemical and hematological parameters. The cytotoxic study was conducted by the MTT method. The histopathological study was conducted in brain and liver tissues. Results: Acute treatment with TG1 (dose of 30 mg. kg -1) did not change the general behavior pattern of rats and not result in hematological and histological changes in the liver. The reduced levels of transaminase and alkaline phosphatase (ALKP) enzymes may suggest even certain liver protection. The biochemical analyzes demonstrated low levels of aspartate aminotransferase, ALKP and uric acid, providing preservation of kidneys and livers of animals (p<0.05). TG1 this study did not reveal cytotoxic potential by MTT method. Conclusion: These results indicate that treatment with TG1 not produce hematological, biochemical and histopathological alterations in rats suggesting low toxicity

Pharmacological evidence of α2-adrenergic receptors in the hypotensive effect of Platonia insignis mart.

Platonia insignis Mart. (Clusiaceae) is a medicinal plant from the Brazilian Amazon region. The present study evaluated the biological potential of the ethanol extract (Pi-EtOH) and ethyl acetate fraction (Pi-EtOAc) of the P. insignis fruit shells on the cardiovascular system of rats. Pi-EtOH or Pi-EtOAc (12.5, 25, and 50 mg/kg) was administered intravenously in normotensive rats (260-300 g), and the mean arterial pressure and the heart rate were monitored. The Pi-EtOH induced hypotension (-11.56±0.89, -7.43±0.85, and -17.56±1.97 mmHg) followed by bradycardia in two highest doses (-8.89±3.62 and -15.79±1.83 beats/min) and Pi-EtOAc, at the same doses, induced hypotension (-11.2±1.03, -14.48±1.13, -29.89±2.67 mmHg) more intensively, followed by tachycardia at the dose 12.5 and 25 mg/kg (15.64±2.06, 19.31±1.92 beats/min) and bradycardia at a dose of 50 mg/kg (-9.98±7.33 beats/min). The hypotensive response from Pi-EtOAc was not attenuated when used in the pretreatment with L-NAME, verapamil, propranolol, and hexamethonium. However, when using yohimbine, the hypotensive effect was inhibited (-4.42±1.28 (P<.05), -3.29±0.99 (P<.05), 2.06±1.18 mmHg (P<.05); Student's t-test). Hence, the Pi-EtOAc seems to act similarly to the α2-adrenergic agonist in this hypotensive effect.

Caracterização anatômica foliar de espécimes de Platonia insignis Mart. (Clusiaceae) em diferentes períodos sazonais / Characterization of anatomical specimens of leaf Platonia insignis Mart. (Clusiaceae) in different seasonal periods

Platonia insignis Mart. (Clusiaceae) é conhecida na Amazônia brasileira como "bacuri" e apresenta grande importância econômica no Estado do Pará, especialmente na zona do Salgado, onde o extrativismo vegetal é considerado uma das principais fontes de renda da população. Para complementar o conhecimento biológico da espécie, foi realizado o estudo de caracterização anatômica das folhas de quinze espécimes procedentes de três áreas de floresta secundária do nordeste do Pará, Município de Bragança, na localidade de Benjamin Constant; do Município de São Caetano de Odivelas e da ilha do estuário guajarino de Mosqueiro. Foram aplicadas técnicas histológicas de dissociação de epidermes, secções histológicas, testes microquímicos e análise em microscopia eletrônica de varredura. Os resultados mostram cutícula espessa, cera epicuticular em crosta, raros tricomas, folhas anfiestomáticas com estômatos paracíticos, presença de colênquima angular e de estruturas secretoras. No estudo não foram observadas diferenças na estrutura foliar dos espécimes, porém foi constatada a existência de plasticidade fenotípica.

Platonia insignis Mart. is known in the Brazilian Amazon as "bacuri" and has great economic importance in the State of Pará, especially in the zona do Salgado, where the extraction of plants is considered a major source of income to the population. To complement the biological knowledge of the species, the study was conducted to characterize leaf anatomical fifteen specimens from three areas of secondary forest in northeastern Pará, Municipality of Bragança, in the town of Benjamin Constant, in São Caetano de Odivelas island estuary Guajarina Mosqueiro. Histological techniques were applied to dissociation of epidermis, the histological sections, microchemical tests and analysis in scanning electron microscopy. The results show a thick cuticle, epicuticular wax crust, rare trichomes, leaves with amphistomatic paracytic stomata, presence of collenchyma and secretory structures. In the study there were no differences in leaf structure of the specimens, however, was found to contain phenotypic plasticity.

MORFOLOGIA DOS FRUTOS, SEMENTES E PLÂNTULAS DE Platonia insigms MART. (CLUSIACEAE). I. ASPECTOS ANATÔMICOS DOS FRUTOS E SEMENTES EM DESENVOLVIMENTO

Morphological, structural and developmental features of fruits and seeds of Platonia insignis(Clusiaceae) are described, with the purpose of elucidating the origin of the pulp present in the fruit. The pulp, which involves the seeds, emerges early in fruit development. The sept and the inner endocarp cell layers undergo accentuated radial elongation and become firmly attached to the testai outer layers. At maturation the endocarp is released from the pericarp, remaining attached to the seeds.

São descritos aspectos morfológicos e estruturais dos frutos e sementes, em desenvolvimento, de Platonia insignis(Clusiaceae), visando principalmente esclarecer a origem da camada comestível presente no fruto. Concluiu-se que a camada carnosa branco-amarelada, que envolve as sementes, é de origem endocárpica e começa a se diferenciar já nos estádios iniciais do desenvolvimento do fruto, através de um acentuado alongamento radial das camadas mais internas do endocarpo. Estas, juntamente com as células dos septos, que também se alongam à medida que o fruto se desenvolve, vão se aderindo à testa por meio de interdigitações. Posteriormente, no fruto maduro, o endocarpo destaca-se do restante do pericarpo, permanecendo firmemente ligado à semente.

MORFOLOGIA DOS FRUTOS, SEMENTES E PLÂNTULAS DE Platonia insignis MART. (CLUSIACEAE). II. MORFO-ANATOMIA DOS FRUTOS E SEMENTES MADUROS

The fruit of Platonia insignisis an unilocular berry; oblate to oblong in shape. The exocarp is uniseriate; the mesocarp is parenchymatic, with branched secretory ducts and vascular bundles. At maturation, the endocarp constitutes the pulpy layer which involves the seed, when it is released from the rest of the pericarp; the inner endocarp cell layers are radially elongated and remain firmly attached to the testai outer layers. The mature seeds are anatropous, becoming unitegmic and exalbuminous; the testa is multiseriate and shows a single rapheal vascular bundle with many postchalazal branches; the tegmen is almost completly crushed, showing only some scattered brachisclereids. The conferruminate embryo is constituted by a thick radicle-hypocotyl axis which is rich in lipids. Secretory ducts occur in the fundamental cortical mer-istem of the embrionary axis.

O fruto de Platonia insignisé uma baga uniloculada cuja forma varia de oblata a oblonga. O exocarpo é unisseriado; o mesocarpo é parenquimático, contendo numerosos dutos secretores e feixes vasculares ramificados; o endocarpo constitui a camada pulposa que envolve as sementes no fruto maduro, quando se destaca do restante do pericarpo. As camadas celulares mais internas do endocarpo são radialmente alongadas e permanecem firmemente aderidas às camadas mais externas da testa, por meio de interdigitações. As sementes são anátropas e se tornam unitegumentadas e exalbuminosas. A testa é multisseriada e apresenta numerosos feixes vasculares que partem do feixe rafeal único como ramos pós-calazais; o tégmen encontra-se colapsado, sendo distintos apenas alguns braquisclereídes dispersos. O embrião, do tipo conferruminado, consta de um espesso eixo hipocótilo-radícula, rico em reservas lipídicas, em cujo meristema fundamental estão presentes abundantes dutos secretores.

MORFOLOGIA DOS FRUTOS, SEMENTES E PLÂNTULAS DE Platonici insignis MART. (CLUSIACEAE). III GERMINAÇÃO E PLÂNTULAS

Morphological and developmental features of seedlings of Platonia insignisMart, are here described. The seedling is cryptocotylar, hypogeal. The primary root is axial; opposite scale leaves (cataphylls) precede the foliage leaves (metaphylls) in the epicotyl. The metaphylls are simple, eliptic, decussate, short petiolate; venation pattern is pinnate, camptodromous, and strongly brochidodromous.

São apresentados aspectos morfológicos das plântulas em desenvolvimento de Platonici insignisMart.. Apiàntala é criptocotiledonar, hipógea A raiz é axial e no epicótilo, desenvolvem-se 2-5 pares de catáfilos opostos, antes do aparecimento do primeiro par de metáfilos. Estes são simples, de disposição oposta cruzada, elípticos, tendo pecíolos curtos, com duas minúsculas alas; a venação é pinada, camptódroma, fortemente broquidódroma.