Characterization of Planktochlorella nurekis Extracts and Virucidal Activity against a Coronavirus Model, the Murine Coronavirus 3

Certain members of the Coronaviridae family have emerged as zoonotic agents and have recently caused severe respiratory diseases in humans and animals, such as SARS, MERS, and, more recently, COVID-19. Antivirals (drugs and antiseptics) capable of controlling viruses at the site of infection are scarce. Microalgae from the Chlorellaceae family are sources of bioactive compounds with antioxidant, antiviral, and antitumor activity. In the present study, we aimed to evaluate various extracts from Planktochlorella nurekis in vitro against murine coronavirus-3 (MHV-3), which is an essential human coronavirus surrogate for laboratory assays. Methanol, hexane, and dichloromethane extracts of P. nurekis were tested in cells infected with MHV-3, and characterized by UV-vis spectrophotometry, nuclear magnetic resonance (NMR) spectroscopy, ultraperformance liquid chromatography-mass spectrometry (UPLC-MS), and the application of chemometrics through principal component analysis (PCA). All the extracts were highly efficient against MHV-3 (more than a 6 Log unit reduction), regardless of the solvent used or the concentration of the extract, but the dichloromethane extract was the most effective. Chemical characterization by spectrophotometry and NMR, with the aid of statistical analysis, showed that polyphenols, carbohydrates, and isoprene derivatives, such as terpenes and carotenoids have a more significant impact on the virucidal potential. Compounds identified by UPLC-MS were mainly lipids and only found in the dichloromethane extract. These results open new biotechnological possibilities to explore the biomass of P. nurekis; it is a natural extract and shows low cytotoxicity and an excellent antiviral effect, with low production costs, highlighting a promising potential for development and implementation of therapies against coronaviruses, such as SARS-CoV-2.This research was funded LVA-MIP-CCB-UFSC/Sigpex: 201917940, and CNPq, CAPES-DS

Estudo fitoquímico e propriedades biológicas de espécies de Polygala (Polygalaceae)

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Físicas e Matemáticas, Programa de Pós-Graduação em Química, 2019.As espécies do gênero Polygala, um dos 19 gêneros da família Polygalaceae, com cerca de 725 espécies, são herbáceas e algumas espécies possuem aroma característico de salicilato de metila em suas raízes. As xantonas constituem-se nos principais metabólitos secundários encontrados em espécies de Polygala, mas que contém ainda saponinas triterpênicas, cumarinas, esterois, flavonoides e, mais raramente, estirilpironas. Em função de usos na medicina tradicional para o tratamento de contusões e processos inflamatórios, diversos estudos descreveram as atividades farmacológicas centrais e modelos anti-inflamatórios para suas substâncias. As espécies P. altomontana, P. densiracemosa e P. lancifolia (Polygalaceae), por carecerem de estudos, foram submetidas a um estudo fitoquímico e biológico. As principais substâncias foram purificadas a partir de extratos brutos por métodos de fracionamentos cromatográficos e cristalizações. As atividades biológicas envolveram modelos in vitro e in vivo para se caracterizar suas propriedades, além de outras relacionadas ao processo ADME. Da espécie P. altomontana foram isolados 9 compostos, todos novos na espécie, sendo uma cumarina, um esterol, uma di-hidroestirilpirona, quatro estirilpironas e dois flavonois, dos quais uma estirilpirona inédita e um flavonoide isolado pela primeira vez no gênero. As espécies P. densiracemosa e P. lancifolia resultaram no isolamento de um esterol e duas xantonas para cada espécie, sendo duas xantonas inéditas enquanto que, para P. altomontana, foram isoladas di-hidro e estirilpironas ao invés de xantonas. A cumarina e os dois flavonoides de P. altomontana foram quantificados por EC-DAD. A di-hidroestirilpirona, uma estirilpirona e uma xantona avaliadas por ensaios de permeabilidade, PAMPA TGI e PAMPA BHE, indicaram melhores resultados para a di-hidroestirilpirona. O esterol, a cumarina e uma xantona apresentaram log P > 5, enquanto os flavonoides glicosilados apresentaram log P negativos, coerentes com melhor lipofilicidade ou hidrofilicidade para suas estruturas. O extrato de P. altomontana apresentou os melhores índices de proteção antioxidante, correlacionados com o conteúdo de fenólicos nos métodos com DPPH e ABTS. Nos ensaios de atividade antimicrobiana, a fração acetato de etila do extrato de P. altomontana demonstrou atividade moderada (CIM de 500 µg mL-1) contra as bactérias S. aureus e P. aeruginosa e CIM de 125 µg mL-1 contra os fungos C. gattii e C. neoformans. O extrato bruto de P. densiracemosa apresentou inibição moderada contra células de leucemia mieloide (66% para HL-60, 52% para Jurkat), com baixa toxicidade (12% contra células normais Vero). Nos ensaios pré-clínicos de atividade farmacológica central, extratos da espécie P. altomontana mostraram consistentes efeitos ansiolíticos, hipnosedativos, anticonvulsivantes, bem como reversão do efeito do comprometimento da memória induzido por STZ em ratos. Nos ensaios pré-clínicos de efeitos antinociceptivo e anti-inflamatório os extratos brutos de P. altomontana, nas doses de 30, 100, 300 e 1000 mg/kg, causaram inibição significativa da resposta nociceptiva na fase inflamatória.Abstract : The species of the genus Polygala, one of the 19 genus of the Polygalaceae family, with approximately 725 species, are herbaceous and some species have a characteristic aroma of methyl salicylate in their roots. The xanthones are the major secondary metabolites found in species of Polygala, but they still contain triterpenic saponins, coumarins, sterols, flavonoids, and more rarely, styryl-pyrones. Due to its uses in traditional medicine for the treatment of contusions and inflammatory processes, several studies have described the central pharmacological activities and anti-inflammatory models for its substances. The species P. altomontana, P. densiracemosa and P. lancifolia (Polygalaceae), due to lack of studies, were submitted to phytochemical and biological studies. The major substances were purified from crude extracts by chromatographic fractionation and crystallization methods. The biological activities involved in vitro and in vivo models to characterize their properties, in addition to others related to the ADME process. In the P. altomontana species, nine compounds were isolated, all new to the species, being one coumarin, one sterol, one dihydrostyryl-pyrone, four styryl-pyrones and two flavonoids, of which one styryl-pyrone is unpublished and one flavonoid was isolated for the first time in the genus. The species P. densiracemosa and P. lancifolia resulted in the isolation of one sterol and two xanthones for each species, being two unpublished xanthones, whereas for P. altomontana, dihydro and styryl-pyrones were isolated instead of xanthones. The coumarin and the two flavonoids from P. altomontana were quantified by CE-DAD. The dihydrostyryl-pyrone, one styryl-pyrone and one xanthone evaluated by permeability assays, PAMPA TGI and PAMPA BHE, indicated better results for dihydrostyryl-pyrone. The sterol, the coumarin and one xanthone presented log P> 5, while the glycosylated flavonoids presented negative log P, consistent with better lipophilicity or hydrophilicity for their structures. The extract of P. altomontana showed the best antioxidant protection index, correlated with phenolic content in the methods with DPPH and ABTS. In the antimicrobial activity assays, the ethyl acetate fraction of the P. altomontana extract showed moderate activity (MIC of 500 µg mL-1) against S. aureus and P. aeruginosa and MIC of 125 µg mL-1 against fungi C. gattii and C. neoformans. The crude extract of P. densiracemosa showed moderate inhibition against myeloid leukemia cells (66% for HL-60, 52% for Jurkat), with low toxicity (12% against normal Vero cells). In the pre-clinical studies of central pharmacological activity, extracts of the P. altomontana species showed consistent anxiolytic, hypnosedative and anticonvulsant effects, as well as reversion of the effect of memory impairment induced by STZ in rats. In pre-clinical trials with antinociceptive and anti-inflammatory effects, the crude extracts of P. altomontana at doses of 30, 100, 300 and 1000 mg/kg caused significant inhibition of the nociceptive response in the inflammatory phase