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In a search for natural compounds with anti-HIV-1 activity, we studied the effect of the ethanolic extract obtained from leaves, bark, and peels of Punica granatum L. for the inhibition of the HIV-1 reverse transcriptase (RT)-associated ribonuclease H (RNase H) and integrase (IN) LEDGF-dependent activities. The chemical analyses led to the detection of compounds belonging mainly to the phenolic and flavonoid chemical classes. Ellagic acid, flavones, and triterpenoid molecules were identified in leaves. The bark and peels were characterized by the presence of hydrolyzable tannins, such as punicalins and punicalagins, together with ellagic acid. Among the isolated compounds, the hydrolyzable tannins and ellagic acid showed a very high inhibition (IC50 values ranging from 0.12 to 1.4 µM and 0.065 to 0.09 µM of the RNase H and IN activities, respectively). Of the flavonoids, luteolin and apigenin were found to be able to inhibit RNase H and IN functions (IC50 values in the 3.7–22 μM range), whereas luteolin 7-O-glucoside showed selective activity for HIV-1 IN. In contrast, betulinic acid, ursolic acid, and oleanolic acid were selective for the HIV-1 RNase H activity. Our results strongly support the potential of non-edible P. granatum organs as a valuable source of anti-HIV-1 compounds.

Zika virus, an arthropod-borne flavivirus, is an emerging healthcare threat worldwide. Zika virus is responsible for severe neurological effects, such as paralytic Guillain-Barrè syndrome, in adults, and also congenital malformations, especially microcephaly. No specific antiviral drugs and vaccines are currently available, and treatments are palliative, but medicinal plants show great potential as natural sources of anti-Zika phytochemicals. This study deals with the investigation of the composition, cytotoxicity, and anti-Zika activity of Punica granatum leaf ethanolic extract, fractions, and phytoconstituents. P. granatum leaves were collected from different areas in Italy and Greece in different seasons. Crude extracts were analyzed and fractionated, and the pure compounds were isolated. The phytochemical and biomolecular fingerprint of the pomegranate leaves was determined. The antiviral activities of the leaf extract, fractions, and compounds were investigated against the MR766 and HPF2013 Zika virus strains in vitro. Both the extract and its fractions were found to be active against Zika virus infection. Of the compounds isolated, ellagic acid showed particular anti-Zika activities, with EC50 values of 30.86 µM for MR766 and 46.23 µM for HPF2013. The mechanism of action was investigated using specific antiviral assays, and it was demonstrated that ellagic acid was primarily active as it prevented Zika virus infection and was able to significantly reduce Zika virus progeny production. Our data demonstrate the anti-Zika activity of pomegranate leaf extract and ellagic acid for the first time. These findings identify ellagic acid as a possible anti-Zika candidate compound that can be used for preventive and therapeutic interventions.

Vitis vinifera L. (Vitaceae family) is a popular woody perennial plant commonly used in the winemaking. Unfortunately, viticulture generates huge amounts of residues (i.e. leaves, twigs, seeds, skins), which can cause environmental issues when discarded in open areas. It is therefore extremely important to evaluate the potential reuse of these matrices, to reduce the amount of waste at landfill sites [1],[2]. Among the main grapevine by-products, no data on the green pruning residues (GPRs) produced by the annual spring pruning have been reported. Thus, the aim of this work was the analysis and valorization of GPRs from 16 red and white V. vinifera cultivars from Piedmont (Italy), by comparing the results with those obtained from the leaves of the same cultivars. An experimental design optimization was performed and the obtained hydroalcoholic extracts where then quali- and quantitatively analyzed by HPLC-PDA-MS/MS, with similar polyphenolic profiles for both GPRs and leaves. Quercetin 3-O-glucuronide, caftaric acid and quercetin 3-O-glucoside were the main components of the investigated matrices, although in variable proportions. Considering the prevalent presence of polyphenols in the analyzed extracts their antioxidant activity was evaluated with colorimetric in-vitro assays, off-line combined with HPLC-PDA analysis. In this way the contribution of each compound to the antioxidant activity, in terms of radical scavenging abilities, was determined. These findings could suggest GPRs as a potential source of natural compounds, and therefore promote their use in the food field, as food supplements, which could increase their economic value together with a positive effect on the environment.

A correct botanical identification and analytical quality control of volatile key-markers responsible for aroma and biological activities is necessary to monitor volatile compounds transferred from a plant to the related herbal tea and human intake to guarantee their safe use. This is mainly true for markers limited by regulations or by a recommended maximum amount of consumption per day. GC–MS is the elective technique to analyze volatiles, provided that for aqueous samples (herbal teas) an appropriate sample preparation procedure, and/or a water-compatible GC stationary phases are applied. Solid Phase Micro Extraction (SPME) on-line coupled to GC–MS in a fully automatic approach is here applied to sample and quantify key markers in plant material (headspace) and in the corresponding herbal tea (direct immersion). In parallel, a new generation of GC columns coated with ionic liquid based stationary phases compatible with aqueous samples (Watercol™) was applied to test direct injection of aqueous samples (DAI-GC-FID). The latter approach fully bypasses sample preparation thus speeding up quality control. This study deals with the quantitation of menthol, α- and β-thujone, estragole, and anethole contained in several plant species commonly used for herbal teas (i.e. peppermint, sage, wormwood, fennel, aniseed) and regulated by International Organizations. The two methods gave comparable results and are characterized by high repeatability, linearity and accuracy, although, as expected, their sensitivity was different because DAI-GC-FID implies injection of the sample as such without analyte concentration as for DI-SPME-GC–MS. For instance, LOD and LOQ of estragole were 0.03 and 0.1 mg L−1 with DI-SPME-GC–MS and 0.1 and 0.8 mg L−1 with DAI-GC-FID. The two methods are fully complementary and their adoption depends on the amount of marker(s) to be quantified.