Anti-Zika Virus Activity of Plant Extracts Containing Polyphenols and Triterpenes on Vero CCL-81 and Human Neuroblastoma SH-SY5Y Cells.

Zika virus (ZIKV) infection is a global threat associated to neurological disorders in adults and microcephaly in children born to infected mothers. No vaccine or drug is available against ZIKV. We herein report the anti-ZIKV activity of 36 plant extracts containing polyphenols and/or triterpenes. ZIKV-infected Vero CCL-81 cells were treated with samples at non-cytotoxic concentrations, determined by MTT and LDH assays. One third of the extracts elicited concentration-dependent anti-ZIKV effect, with viral loads reduction from 0.4 to 3.8 log units. The 12 active extracts were tested on ZIKV-infected SH-SY5Y cells and significant reductions of viral loads (in log units) were induced by Maytenus ilicifolia (4.5 log), Terminalia phaeocarpa (3.7 log), Maytenus rigida (1.7 log) and Echinodorus grandiflorus (1.7 log) extracts. Median cytotoxic concentration (CC50 ) of these extracts in Vero cells were higher than in SH-SY5Y lineage. M. ilicifolia (IC50 =16.8±10.3 µg/mL, SI=3.4) and T. phaeocarpa (IC50 =22.0±6.8 µg/mL, SI=4.8) were the most active extracts. UPLC-ESI-MS/MS analysis of M. ilicifolia extract led to the identification of 7 triterpenes, of which lupeol and a mixture of friedelin/friedelinol showed no activity against ZIKV. The composition of T. phaeocarpa extract comprises phenolic acids, ellagitannins and flavonoids, as recently reported by us. In conclusion, the anti-ZIKV activity of 12 plant extracts is here described for the first time and polyphenols and triterpenes were identified as the probable bioactive constituents of T. phaeocarpa and M. ilicifolia, respectively.

Effect of Essential Oils on the Release of TNF-α and CCL2 by LPS-Stimulated THP­1 Cells.

Plants and their constituents have been used to treat diverse ailments since time immemorial. Many plants are used in diverse external and internal formulations (infusions, alcoholic extracts, essential oils (EOs), etc.) in the treatment of inflammation-associated diseases, such as those affecting the respiratory tract or causing gastrointestinal or joint problems, among others. To support the traditional uses of plant extracts, EOs have been assessed for their alleged anti-inflammatory properties. However, the effect of EOs on the release of cytokines and chemokines has been much less reported. Considering their traditional use and commercial relevance in Portugal and Angola, this study evaluated the effect of EOs on the in vitro inhibition of the cytokine tumor necrosis factor-α (TNF-α) and the chemokine (C-C motif) ligand 2 (CCL2) by lipopolysaccharide (LPS)-stimulated human acute monocytic leukemia cells (THP-1 cells). Twenty EOs extracted from eighteen species from seven families, namely from Amaranthaceae (Dysphania ambrosioides), Apiaceae (Foeniculum vulgare), Asteraceae (Brachylaena huillensis, Solidago virgaurea), Euphorbiaceae (Spirostachys africana), Lamiaceae (Lavandula luisieri, Mentha cervina, Origanum majorana, Satureja montana, Thymbra capitata, Thymus mastichina, Thymus vulgaris, Thymus zygis subsp. zygis), Myrtaceae (Eucalyptus globulus subsp. maidenii, Eucalyptus radiata, Eucalyptus viminalis) and Pinaceae (Pinus pinaster) were assayed for the release of CCL2 and TNF-α by LPS-stimulated THP-1 cells. B. huillensis, S. africana, S. montana, Th. mastichina and Th. vulgaris EOs showed toxicity to THP-1 cells, at the lowest concentration tested (10 µg/mL), using the tetrazolium dye assay. The most active EOs in reducing TNF-α release by LPS-stimulated THP-1 cells were those of T. capitata (51% inhibition at 20 µg/mL) and L. luisieri (15-23% inhibition at 30 µg/mL and 78-83% inhibition at 90 µg/mL). L. luisieri EO induced a concentration-dependent inhibition of CCL2 release by LPS­stimulated THP-1 cells (23%, 54% and 82% inhibition at 10, 30 and 90 µg/mL, respectively). These EOs are potentially useful in the management of inflammatory diseases mediated by CCL2 and TNF­α, such as atherosclerosis and arthritis.

Semisynthetic Cardenolides Acting as Antiviral Inhibitors of Influenza A Virus Replication by Preventing Polymerase Complex Formation.

Influenza virus infections represent a major public health issue by causing annual epidemics and occasional pandemics that affect thousands of people worldwide. Vaccination is the main prophylaxis to prevent these epidemics/pandemics, although the effectiveness of licensed vaccines is rather limited due to the constant mutations of influenza virus antigenic characteristics. The available anti-influenza drugs are still restricted and there is an increasing viral resistance to these compounds, thus highlighting the need for research and development of new antiviral drugs. In this work, two semisynthetic derivatives of digitoxigenin, namely C10 (3ß-((N-(2-hydroxyethyl)aminoacetyl)amino-3-deoxydigitoxigenin) and C11 (3ß-(hydroxyacetyl)amino-3-deoxydigitoxigenin), showed anti-influenza A virus activity by affecting the expression of viral proteins at the early and late stages of replication cycle, and altering the transcription and synthesis of new viral proteins, thereby inhibiting the formation of new virions. Such antiviral action occurred due to the interference in the assembly of viral polymerase, resulting in an impaired polymerase activity and, therefore, reducing viral replication. Confirming the in vitro results, a clinically relevant ex vivo model of influenza virus infection of human tumor-free lung tissues corroborated the potential of these compounds, especially C10, to completely abrogate influenza A virus replication at the highest concentration tested (2.0 µM). Taken together, these promising results demonstrated that C10 and C11 can be considered as potential new anti-influenza drug candidates.

Encapsulation of trans-aconitic acid in mucoadhesive microspheres prolongs the anti-inflammatory effect in LPS-induced acute arthritis.

trans-Aconitic acid (TAA) is the main constituent of the leaves from the medicinal plant Echinodorus grandiflorus, used to treat different inflammatory diseases. TAA induces a potent but short-lasting biological response, credited to its high polarity and unfavorable pharmacokinetics. Here we developed, characterized and evaluated the anti-inflammatory activity of mucoadhesive microspheres loaded with TAA. Seven batches of mucoadhesive microspheres were prepared by the emulsification/solvent evaporation method, employing different proportions of TAA and Carbopol 934 or/and hydroxypropylmethylcellulose. All batches were characterized for their particle medium size, polydispersity index and entrapment percentage. The batch coded F3c showed highest entrapment percentage and was characterized by infrared spectroscopy (ATR-FTIR), scanning electron microscopy (SEM), differential scanning calorimetry (DSC), thermogravimetric analyses (TGA) and zeta potential. The anti-inflammatory activity of F3c was assessed in a model of acute arthritis induced by injection of LPS in the knee joint of Swiss mice. The granulometric analyses indicated heterogeneous size distribution for F3c. SEM characterization indicated microspheres with slightly irregular shape and rough surface. Results from ATR-FTIR and thermal analyses (DSC and TGA) pointed out absence of incompatibility between the components of the formulation; thermal events related to the constituents were isolated and randomly located, suggesting amorphous distribution of TAA in the formulation matrix. The zeta potential of the formulations varied from -30 to -34 mV, which may contribute to good stability. When given orally to mice, F3c induced a prolonged anti-inflammatory response by reducing total cell count and neutrophilic accumulation in the joint cavity even when given 48 and 36 h before the stimulus, respectively, in comparison to free TAA (up to 24 and 6 h, respectively). Therefore, the encapsulation of TAA in mucoadhesive microspheres provided its sustained release, indicating that this drug delivery system is a potential agent to treat inflammatory diseases by regulating cell influx.