Ecklonia cava is edible seaweed that is found in Asian countries, such as Japan and Korea; and, its major components include fucoidan and phlorotannins. Phlorotannins that are isolated from E. cava are well-known to have an antioxidant effect and strong antiviral activity against porcine epidemic diarrhea virus (PEDV), which has a high mortality rate in piglets. In this study, the bioactive components were determined based on two different approaches: (i) bio-guided isolation using the antiviral activity against the H1N1 viral strain, which is a representative influenza virus that originates from swine and (ii) high-resolution mass spectrometry-based dereplication, including relative mass defects (RMDs) and HPLC-qTOFMS fragmentation analysis. The EC70 fraction showed the strongest antiviral activity and contained thirteen phlorotannins, which were predicted by dereplication. Ten compounds were directly isolated from E. cava extract and then identified. Moreover, the dereplication method allowed for the discovery of two new phlorotannins. The structures of these two isolated compounds were elucidated using NMR techniques and HPLC-qTOFMS fragmentation analysis. In addition, molecular modelling was applied to determine the absolute configurations of the two new compounds. The antiviral activities of seven major phlorotannins in active fraction were evaluated against two influenza A viral strains (H1N1 and H9N2). Six of the compounds showed moderate to strong effects on both of the viruses and phlorofucofuroeckol A (12), which showed an EC50 value of 13.48 ± 1.93 μM, is a potential active antiviral component of E. cava.

Pinus densiflora was screened in an ongoing project to discover anti-influenza candidates from natural products. An extensive phytochemical investigation provided 26 compounds, including two new megastigmane glycosides (1 and 2), 21 diterpenoids (3–23), and three flavonoids (24–26). The chemical structures were elucidated by a series of chemical reactions, including modified Mosher’s analysis and various spectroscopic measurements such as LC/MS and 1D- and 2D-NMR. The anti-influenza A activities of all isolates were screened by cytopathic effect (CPE) inhibition assays and neuraminidase (NA) inhibition assays. Ten candidates were selected, and detailed mechanistic studies were performed by various assays, such as Western blot, immunofluorescence, real-time PCR and flow cytometry. Compound 5 exerted its antiviral activity not by direct neutralizing virion surface proteins, such as HA, but by inhibiting the expression of viral mRNA. In contrast, compound 24 showed NA inhibitory activity in a noncompetitive manner with little effect on viral mRNA expression. Interestingly, both compounds 5 and 24 were shown to inhibit nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in a dose-dependent manner. Taken together, these results provide not only the chemical profiling of P. densiflora but also anti-influenza A candidates.

In the search for antiviral cyclopeptides against influenza A virus, five unprecedented Caryophyllaceae-type cyclopeptides (1–5) were isolated from the leaves of Melicope pteleifolia. Their chemical structures and absolute configurations were unambiguously determined by means of advanced Marfey’s analysis and comprehensive spectroscopic analyses including two-dimensional nuclear magnetic resonance and MS/MS fragmentation. Interestingly, compounds 3–5 contain an unusual heterocycle, a 3a-hydroxypyrroloindole moiety, which was biosynthetically formed by a nucleophilic cyclization from the least abundant amino acid, tryptophan, precursor and has aroused a great interest in the aspect of chemical diversity and biological activity. All isolates (1–5) were evaluated for their protective effects against influenza A viruses H1N1 and H9N2 in MDCK cells. All isolated cyclopeptides exhibited strong anti-influenza activity, especially against H1N1. Compound 3 showed the most potent CPE inhibition effect, which was stronger than that of the positive control ribavirin against H1N1, with an EC50 (μM) of 2.57 ± 0.45 along with higher selectivity.

Nephelium lappaceum (rambutan) is an edible tropical fruit that is widely grown in Southeast Asia. In general, the seeds contain high nutrients, but rambutan seeds are thrown out during processing. In this study, the anti-aging activity of rambutan seeds was evaluated with a new approach through the selective inhibition of the senescence-associated secretory phenotype (senomorphics). Luciferase promoter assays using p16INK4A and SA-β-gal promoters for rambutan showed that its seeds possessed strong senomorphic activity. Molecular networking using ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-qTOF-MS) with a tandem database (UPLC-qTOF-MS/MS) was applied to determine the chemical composition of rambutan. Based on the activity results, nine compounds, one new (7) and eight known kaempferol type compounds, were isolated from the seeds. Compounds 2, 4 and 9 significantly reduced the mRNA expression levels of senescence markers, such as p16INK4A, p21CIP1, p53 and SA-β-gal. These compounds also significantly increased the level of SIRT1, a longevity modulator. Compounds 2, 4 and 9 decreased the mRNA expression levels of senescence-associated secretory phenotypes (SASPs) and subsequently decreased the number of SA-β-gal-positive cells. Thus, rambutan seeds and its constituents might be able to protect against age-related problems.

During an effort to find insulin mimetic compounds, the leaves of Gymnema inodorum were shown to have a stimulatory effect on glucose uptake in 3T3-L1 adipocyte cells. Bioassay-guided fractionation on a 70% ethanol extract of G. inodorum was applied to yield two new (1 and 2) and two known (8 and 9) oleanane triterpenoids with a methyl anthranilate moiety together with five further new oleanane triterpenoids (3–7). The chemical structures of all isolates were determined based on their spectroscopic data, including IR, UV, NMR, and mass spectrometric analysis. The isolated compounds (1–9) were determined for their stimulatory activities on glucose uptake in differentiated 3T3-L1 adipocyte cells using 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)­amino]-d-glucose (2-NBDG) as a fluorescent-tagged glucose probe. Three compounds (3, 5, and 9) showed stimulatory effects on the uptake of 2-NBDG in 3T3-L1 adipocyte cells. Chemicals with a methyl anthranilate moiety have been considered as crucial contributors of flavor odor in foods, and quantitative analysis showed the content of compound 8 to be 0.90 ± 0.01 mg/g of the total extract. These results suggest that the leaves of G. inodorum have the potential to be used as an antidiabetic functional food or tea.

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Alzheimer’s disease (AD), a neurocognitive impairment affecting human mental capacity, is related to the accumulation of amyloid-β peptide (Aβ) and the hyperphosphorylation of tau protein. In addition to modern therapies approved for AD treatment, natural products with antioxidant and anti-inflammatory properties have been studied for their potential to prevent AD pathogenesis. Six new noroleanane triterpenoids from the fruit peels of Camellia japonica were isolated, and their structures were determined by diverse spectroscopic methods. The neuroprotective effects of the six new compounds were tested against Aβ-induced neurotoxicity and neuroinflammation in mouse hippocampal and microglial cells. In the model of HT22-transfected cells, compounds 1–4 showed strongly neuroprotective effects via antioxidant response element gene activation and decreased the level of glutamate uptake. Compounds 1–4 also appeared to have strong inhibitory effects on NO production in Aβ1–42-transfected BV2 microglial cells. A docking simulation study was used to explain the inhibitory effects of compounds 1–4 on β-secretase 1 (BACE1). Noroleanane triterpenoids 1–4 had potential neuroprotective and anti-inflammatory effects against Aβ-induced neuronal damage. The structure–activity relationships of the 30 oleanane triterpenoids from C. japonica were assessed in a model of Aβ1–42-transfected HT22 cells.

Microbial cocultivation has been applied as a strategy to induce the biosynthesis of specialized metabolites. However, most previous studies have focused on competitive interactions between test strains. During our LC-MS-based chemical screening of randomized cocultures of Basidiomycetous fungi, we discovered that the coculture of Phellinus orientoasiaticus (Hymenochaetaceae) and Xylodon f laviporus (Schizoporaceae) induces multiple metabolites, although they did not show any competitive morphology. Targeted isolation yielded three new sesquiterpenes (1−3) along with five known analogues (4−8). The structures of the isolates were determined by MS and NMR experiments as well as electronic circular dichroism analysis. LC-MS analysis suggested that cyclohumulanoids of illudane-, sterpurane-, and tremulane-type scaffolds (1−7) were produced by P. orientoasiaticus, whereas a drimane-type sesquiterpene (8) was produced by X. f laviporus. None of the isolates exhibited antifungal activity or cytotoxicity, and compounds 1−7 exhibited NO production of LPS-treated RAW276.4 cells in a range of 15.9% to 38.0% at 100 μM.