Particulate matter-mediated oxidative stress induces airway inflammation and pulmonary dysfunction through TXNIP/NF-κB and modulation of the SIRT1-mediated p53 and TGF-ß/Smad3 pathways in mice.

Exposure to particulate matter is currently recognized as a serious aggravating factor of respiratory diseases. In this study, we investigated the effects of particulate matter (PM) on the respiratory system in BALB/c mice and NCI-H292 cells. PM (0, 2.5, 5 and 20 mg/kg) was administered to mice by intra-tracheal instillation for 7 days. After a 7 day-repeated treatment of PM, we evaluated inflammatory cytokines/cell counts in bronchoalveolar lavage fluid (BALF) and conducted pulmonary histology and functional test. We also investigated the role of TXNIP/NF-κB and SIRT1-mediated p53 and TGF-ß/Smad3 pathways in PM-induced airway inflammation and pulmonary dysfunction. PM caused a significant increase in pro-inflammatory cytokines, inflammatory cell counts in bronchoalveolar lavage fluid. PM-mediated oxidative stress down-regulated thioredoxin-1 and up-regulated thioredoxin-interacting protein and activation of nuclear factor-kappa B in the lung tissue and PM-treated NCI-H292 cells. PM suppressed sirtuin1 protein levels and increased p53 acetylation in PM-exposed mice and PM-treated NCI-H292 cells. In addition, PM caused inflammatory cell infiltration and the thickening of alveolar walls by exacerbating the inflammatory response in the lung tissue. PM increased levels of transforming growth factor-ß, phosphorylation of Smad3 and activation of α-smooth muscle actin, and collagen type1A2 in PM-exposed mice and PM-treated NCI-H292 cells. In pulmonary function tests, PM exposure impaired pulmonary function resembling pulmonary fibrosis, characterized by increased resistance and elastance of the respiratory system, and resistance, elastance, and damping of lung tissues, whereas decreased compliance of the respiratory system, forced expired volume and forced vital capacity. Overall, PM-mediated oxidative stress caused airway inflammation and pulmonary dysfunction with pulmonary fibrosis via TXNIP pathway/NF-κB activation and modulation of the SIRT1-mediated TGF-ß/Smad3 pathways. The results of this study can provide fundamental data on the potential adverse effects and underlying mechanism of pulmonary fibrosis caused by PM exposure as a public health concern. Due to the potential toxicity of PM, people with respiratory disease must be careful with PM exposure.

Myristica fragrans Extract Inhibits Platelet Desialylation and Activation to Ameliorate Sepsis-Associated Thrombocytopenia in a Murine CLP-Induced Sepsis Model.

Sepsis, characterized by an uncontrolled host inflammatory response to infections, remains a leading cause of death in critically ill patients worldwide. Sepsis-associated thrombocytopenia (SAT), a common disease in patients with sepsis, is an indicator of disease severity. Therefore, alleviating SAT is an important aspect of sepsis treatment; however, platelet transfusion is the only available treatment strategy for SAT. The pathogenesis of SAT involves increased platelet desialylation and activation. In this study, we investigated the effects of Myristica fragrans ethanol extract (MF) on sepsis and SAT. Desialylation and activation of platelets treated with sialidase and adenosine diphosphate (platelet agonist) were assessed using flow cytometry. The extract inhibited platelet desialylation and activation via inhibiting bacterial sialidase activity in washed platelets. Moreover, MF improved survival and reduced organ damage and inflammation in a mouse model of cecal ligation and puncture (CLP)-induced sepsis. It also prevented platelet desialylation and activation via inhibiting circulating sialidase activity, while maintaining platelet count. Inhibition of platelet desialylation reduces hepatic Ashwell-Morell receptor-mediated platelet clearance, thereby reducing hepatic JAK2/STAT3 phosphorylation and thrombopoietin mRNA expression. This study lays a foundation for the development of plant-derived therapeutics for sepsis and SAT and provides insights into sialidase-inhibition-based sepsis treatment strategies.

Antiviral activity of CAVAC-1901, a combination of 3 standardized medicinal plants, against highly pathogenic influenza A virus in chickens.

Three different medicinal plants that consisted of the formulated mixture (CAVAC-1901) have been traditionally used for distinct medicinal purposes in different areas. Angelica dahurica has been used as an important ingredient of a prescription, Gumiganghwal-tang, for the common cold and influenza. Curcuma longa has been utilized for the treatment of asthma, and jaundice. Pinus densiflora (Korean red pine) has been used to improve memory and brain function for the treatment of vascular. Industrial livestock, which are characterized by dense breeding, are vulnerable to influenza infection, causing severe economic loss and social problems. However, there are no viable alternatives due to the risk of the occurrence of variants. Therefore, the aim of this study was to discover anti-influenza combinations of different medicinal plants with the concept of a multicomponent and multitarget (MCMT) strategy in traditional Chinese medicine (TCM). As part of a continuous project, 3 medicinal plants whose inhibitory activity against influenza A was previously reported at the compound level, and the inhibition of cytopathic effects (CPEs) by these formulated mixtures was evaluated against influenza A virus H1N1. A selected combination with an optimal ratio exhibiting synergistic activity was assessed for its antiviral activity in chickens against the highly pathogenic avian influenza (HPAI) H5N6. The selected combination (CAVAC-1901) showed potent inhibitory effects on the expression of neuraminidase and nucleoprotein, by RT-qPCR, Western blot, and immunofluorescence assays. The antiviral activity was more evident in chickens infected with H5N6. The sample-treated group (50 mg/kg/d) decreased mortality and virus titers in various organs. Our results indirectly suggest synergistic inhibitory activity of the combination of 3 different medicinal plants with different modes of action. Taken together, an optimally formulated mixture (CAVAC-1901) could serve as an effective alternative to current measures to minimize damage caused by HPAIs.

Immunological Effects of Aster yomena Callus-Derived Extracellular Vesicles as Potential Therapeutic Agents against Allergic Asthma.

Plant-derived extracellular vesicles, (EVs), have recently gained attention as potential therapeutic candidates. However, the varying properties of plants that are dependent on their growth conditions, and the unsustainable production of plant-derived EVs hinder drug development. Herein, we analyzed the secondary metabolites of Aster yomena callus-derived EVs (AYC-EVs) obtained via plant tissue cultures and performed an immune functional assay to assess the potential therapeutic effects of AYC-EVs against inflammatory diseases. AYC-EVs, approximately 225 nm in size, were isolated using tangential flow filtration (TFF) and cushioned ultracentrifugation. Metabolomic analysis, using ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF/MS), revealed that AYC-EVs contained 17 major metabolites. AYC-EVs inhibited the phenotypic and functional maturation of LPS-treated dendritic cells (DCs). Furthermore, LPS-treated DCs exposed to AYC-EVs showed decreased immunostimulatory capacity during induction of CD4+ and CD8+ T-cell proliferation and activation. AYC-EVs inhibited T-cell reactions associated with the etiology of asthma in asthmatic mouse models and improved various symptoms of asthma. This regulatory effect of AYC-EVs resembled that of dexamethasone, which is currently used to treat inflammatory diseases. These results provide a foundation for the development of plant-derived therapeutic agents for the treatment of various inflammatory diseases, as well as providing an insight into the possible mechanisms of action of AYC-EVs.

Inhibition of a broad range of SARS-CoV-2 variants by antiviral phytochemicals in hACE2 mice.

Although several vaccines and antiviral drugs against SARS-CoV-2 are currently available, control and prevention of COVID-19 through these interventions is limited due to inaccessibility and economic issues in some regions and countries. Moreover, incomplete viral clearance by ineffective therapeutics may lead to rapid genetic evolution, resulting in the emergence of new SARS-CoV-2 variants that may escape the host immune system as well as currently available COVID-19 vaccines. Here, we report that phytochemicals extracted from Chlorella spp. and Psidium guajava possess broad-spectrum antiviral activity against a range of SARS-CoV-2 variants. Through chromatography-based screening, we identified four bioactive compounds and subsequently demonstrated their potential antiviral activities in vivo. Interestingly, in hACE2 mice, treatment with these compounds significantly attenuates SARS-CoV-2-induced proinflammatory responses, demonstrating their potential anti-inflammatory activity. Collectively, our study suggests that phytochemicals from edible plants may be readily available therapeutics and prophylactics against multiple SARS-CoV-2 strains and variants.

Flavonostilbenes Isolated from the Stems of Rhamnoneuron balansae as Potential SIRT1 Activators.

The cumulative effects of cell damage result in aging, which gradually decreases human function in various aspects and leads to multiple age-related chronic diseases. To overcome the adverse effects of aging, silent mating type information regulation 2 homologue (SIRT1) activators are promising bioactive compounds that mimic calorie restriction to improve quality of life and prevent aging. In this study, 11 new flavonostilbenes (1-11) and three known compounds (12-14) were purified from stems of Rhamnoneuron balansae. The structures of the new compounds were determined using extensive data from spectroscopic methods, including NMR and HRESIMS. Their absolute configurations were deduced by ECD calculations with coupling constant analysis. All of the isolated new compounds (1-11) were evaluated for their effects on SIRT1 deacetylase activity, the NAD+/NADH ratio, and the AMP-activated protein kinase activation level in cell-based assays. The results showed that rhamnoneuronal D (1) exhibits promising biological activity in several in vitro models related to SIRT1 and suggest it is a potential natural-product-based antiaging agent.

Rugonidines A-F, Diastereomeric 1,6-Dioxa-7,9-diazaspiro[4.5]dec-7-en-8-amines from the Leaves of Alchornea rugosa.

Rugonidines A-F (1-6), three pairs of novel configurationally semistable diastereomers featuring an unprecedented 1,6-dioxa-7,9-diazaspiro[4.5]dec-7-en-8-amine scaffold, were isolated from Alchornea rugosa based on MS/MS-based molecular networking analysis. Their structures were elucidated by NMR spectroscopy in combination with quantum-chemical calculations. Compounds 1-3 showed a significant increase in glucose uptake level in differentiated 3T3-L1 adipocytes using 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-d-glucose (2-NBDG) as a fluorescent-tagged glucose probe.

Triterpenoid saponins from the leaves and stems of Pericampylus glaucus and their insulin mimetic activities.

During an attempt to discover insulin mimetics, thirteen new triterpenoid saponins (1-13), including three phytolaccagenic acids (1, 2, and 12) and ten serjanic acids (3-11 and 13), as aglycones were isolated from a 70% ethanol extract of leaves and stems from Pericampylus glaucus. The chemical structures of compounds 1-13 were determined through spectroscopic data analysis, including NMR, IR, and HRESIMS. All isolated compounds (1-13) were evaluated using 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-d-glucose (2-NBDG) as a fluorescent-tagged glucose probe to determine their stimulatory effects on glucose uptake in differentiated 3 T3-L1 adipocyte cells. Consequently, four compounds (4, 7, 11, and 12) exhibited stimulatory effects on glucose uptake.

Flavanonol Glycosides from the Stems of Myrsine seguinii and Their Neuroprotective Activities.

The accumulation of amyloid beta (Aß) peptides is common in the brains of patients with Alzheimer's disease, who are characterized by neurological cognitive impairment. In the search for materials with inhibitory activity against the accumulation of the Aß peptide, seven undescribed flavanonol glycosides (1-7) and five known compounds (8-12) were isolated from stems of Myrsine seguinii by HPLC-qTOF MS/MS-based molecular networking. Interestingly, this plant has been used as a folk medicine for the treatment of various inflammatory conditions. The chemical structures of the isolated compounds (1-12) were elucidated based on spectroscopic data, including 1D and 2D nuclear magnetic resonance (NMR), high-resolution electrospray ionization mass spectrometry (HRESIMS) and electronic circular dichroism (ECD) data. Compounds 2, 6 and 7 showed neuroprotective activity against Aß-induced cytotoxicity in Aß42-transfected HT22 cells.

SIRT1 Activation Enhancing 8,3'-Neolignans from the Twigs of Corylopsis coreana Uyeki.

Three undescribed 8,3'-neolignans, corynol (1), 3-methoxy-corynol (2) and 3'-deoxy-corynol (3), together with two bergenin derivatives, three flavonoids, two hydrolysable tannins and six simple phenolic compounds, were isolated from the twigs of Corylopsis coreana Uyeki. The structures of the 8,3'-neolignans were elucidated by analyzing their NMR, HRESIMS and ECD spectra. All the isolated compounds were evaluated for their SIRT1 stimulatory activity, and 3'-deoxy-corynol (3) showed SIRT1 stimulation activity. Furthermore, a docking study of 3 was performed with three representative binding pockets of SIRT1.

Meroterpenoids from the leaves of Psidium guajava (guava) cultivated in Korea using MS/MS-based molecular networking.

MS/MS-based molecular networking showed differences in the chemical profiles, especially the terpenoid-coupled-phloroglucinol clusters, of Psidium guajava grown in Jeju Island of South Korea ("Jejuguava"), Vietnam and China. A chemical investigation of the 95% EtOH extract of Jejuguava leaves revealed meroterpenoids characterized by a dihydropyran ring junction between an acylphloroglucinol structure and terpenoid, and named jejuguajavones A-J (1-10). Compounds (±)-8-(±)-10 are racemic mixtures that were separated using a chiral HPLC column. The chemical structures of all the isolated compounds (1-10) were determined by analyzing the spectroscopic data and performing electronic circular dichroism calculations. Among the isolates, compounds 1-4 exhibit inhibitory activity against the protein tyrosine phosphatase 1B (PTP1B) enzyme, and this result was confirmed by molecular docking simulations.

Alnus hirsuta (Spach) Rupr. Attenuates Airway Inflammation and Mucus Overproduction in a Murine Model of Ovalbumin-Challenged Asthma.

Alnus hirsuta (Spach) Rupr. (AH), a member of the Betulaceae family, is widely used in Eastern Asia of as a source of medicinal compounds for the treatment of hemorrhage, diarrhea, and alcoholism. In this study, we investigated the protective effects of a methanolic extract of AH branches against airway inflammation and mucus production in tumor necrosis factor (TNF)-α-stimulated NCI-H292 cells and in an ovalbumin (OVA)-challenged allergic asthma mouse model. Female BALB/c mice were injected with OVA (40 µg) and aluminum hydroxide (2 mg) on days 0 and 14 to induce allergic airway inflammation. The mice were then challenged with 1% OVA from days 21-23. Mice were treated with AH (50 and 100 mg/kg/day; 2% DMSO) or dexamethasone (positive control; 3 mg/kg/day) from days 18-23. AH treatment effectively attenuated airway resistance/hyperresponsiveness and reduced levels of T helper type 2 (Th2) cytokines, eotaxins, and number of inflammatory cells in bronchoalveolar lavage fluid, and immunoglobulin E in serums of OVA-challenged mice. In histological analysis, AH treatment significantly inhibited airway inflammation and mucus production in OVA-challenged mice. AH treatment downregulated the phosphorylation of I kappa B-alpha, p65 nuclear factor-kappa B (p65NF-κB), and mitogen-activated protein kinases with suppression of mucin 5AC (MUC5AC) in lung tissue. Moreover, AH treatment decreased the levels of pro-inflammatory cytokines and Th2 cytokines, as well as MUC5AC expression, and inhibited the phosphorylation of p65NF-κB in TNF-α-stimulated NCI-H292 cells. These results indicate that AH might represent a useful therapeutic agent for the treatment of allergic asthma.

Melicopteline A-E, Unusual Cyclopeptide Alkaloids with Antiviral Activity against Influenza A Virus from Melicope pteleifolia.

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.

Phenolic Constituents of the Roots of Rhamnoneuron balansae with Senolytic Activity.

With the advent of senolytic agents capable of selectively removing senescent cells in old tissues, the perception of age-associated diseases has been changing from being an inevitable to a preventable phenomenon of human life. In the search for materials with senolytic activity from natural products, six new flavonostilbenes (1-6), three new phenylethylchromanones (7-9), three new phenylethylchromones (10-12), and four known compounds (13-16) were isolated from the roots of Rhamnoneuron balansae. The chemical structures of these isolated compounds were determined based on the interpretation of spectroscopic data, including 1D and 2D NMR, ECD, and HRMS. The absolute configuration of compound 1 was also determined by a Mosher ester analysis and ECD calculations. Compounds 6-8 were shown to selectively destroy senescent cells, and the promoter activity of p16INK4A, a representative senescence marker, was reduced significantly by compound 6. The present results suggest the potential activity of flavonostilbene and phenylethylchromanone skeletons from R. balansae as new senolytics.

Lindera obtusiloba Attenuates Oxidative Stress and Airway Inflammation in a Murine Model of Ovalbumin-Challenged Asthma.

Lindera obtusiloba is widespread in northeast Asia and used for treatment of improvement of blood circulation and anti-inflammation. In this study, we investigated anti-inflammatory and anti-oxidant effects of the methanolic extract of L. obtusiloba leaves (LOL) in an ovalbumin (OVA)-challenged allergic asthma model and tumor necrosis factor (TNF)-α-stimulated NCI-H292 cell. Female BALB/c mice were sensitized with OVA by intraperitoneal injection on days 0 and 14, and airway-challenged with OVA from days 21 to 23. Mice were administered 50 and 100 mg/kg of LOL by oral gavage 1 h before the challenge. LOL treatment effectively decreased airway hyper-responsiveness and inhibited inflammatory cell recruitment, Th2 cytokines, mucin 5AC (MUC5AC) in bronchoalveolar lavage fluid in OVA-challenged mice, which were accompanied by marked suppression of airway inflammation and mucus production in the lung tissue. LOL pretreatment inhibited the phosphorylation of mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-κB) with suppression of activator protein (AP)-1 and MUC5AC in the lung tissue. LOL also down-regulated expression of inflammatory cytokines, and inhibited the activation of NF-κB in TNF-α-stimulated NCI-H292 cells. LOL elevated the translocation of nuclear factor-erythroid 2-related factor (Nrf-2) into nucleus concurrent with increase of heme oxyngenase-1 (HO-1) and NAD(P)H quinine oxidoreductase 1 (NQO1). Moreover, LOL treatment exhibited a marked increase in the anti-oxidant enzymes activities, whereas effectively suppressed the production of reactive oxygen species and nitric oxide, as well as lipid peroxidation in lung tissue of OVA-challenged mice and TNF-α-stimulated NCI-H292 cells. These findings suggest that LOL might serve as a therapeutic agent for the treatment of allergic asthma.

Neuroprotective Effects of Triterpenoids from Camellia japonica against Amyloid ß-Induced Neuronal Damage.

Alzheimer's disease (AD), a neurocognitive impairment affecting human mental capacity, is related to the accumulation of amyloid-ß peptide (Aß) 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 Aß-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 Aß1-42-transfected BV2 microglial cells. A docking simulation study was used to explain the inhibitory effects of compounds 1-4 on ß-secretase 1 (BACE1). Noroleanane triterpenoids 1-4 had potential neuroprotective and anti-inflammatory effects against Aß-induced neuronal damage. The structure-activity relationships of the 30 oleanane triterpenoids from C. japonica were assessed in a model of Aß1-42-transfected HT22 cells.

Metabolite Profiling of Rambutan (Nephelium lappaceum L.) Seeds Using UPLC-qTOF-MS/MS and Senomorphic Effects in Aged Human Dermal Fibroblasts.

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-ß-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-ß-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-ß-gal-positive cells. Thus, rambutan seeds and its constituents might be able to protect against age-related problems.

Antiviral Activities of Compounds Isolated from Pinus densiflora (Pine Tree) against the Influenza A Virus.

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.

Antiviral activity of furanocoumarins isolated from Angelica dahurica against influenza a viruses H1N1 and H9N2.

ETHNOPHARMACOLOGICAL RELEVANCE: Angelica dahurica (Hoffm.) Benth. & Hook.f. ex Franch. & Sav. (Umbelliferae family) is an herbaceous, perennial plant native to northern and eastern Asia. The root of A. dahurica has traditionally been used under the name "Bai Zhi" as a medicinal plant for colds, dizziness, ulcers, and rheumatism. Moreover, it is also an important ingredient of various prescriptions, such as Gumiganghwal-Tang, for the common cold and influenza. AIM OF THE STUDY: Even though various biological activities of the root of A. dahurica have been reported along with its chemical components, the detailed mechanism of how it exerts anti-influenza activity at the compound level has not been studied. Therefore, we investigated the anti-influenza properties of furanocoumarins purified by bioactivity-guided isolation. MATERIALS AND METHODS: Bioactivity-guided isolation from a 70% EtOH extract of the root of A. dahurica was performed to produce four active furanocoumarins. The inhibition of cytopathic effects (CPEs) was evaluated to ascertain the antiviral activity of these compounds against influenza A (H1N1 and H9N2) viruses. The most potent compound was subjected to detailed mechanistic studies such as the inhibition of viral protein synthesis, CPE inhibition in different phases of the viral replication cycle, neuraminidase (NA) inhibition, antiapoptotic activity using flow cytometry, and immunofluorescence. RESULTS: The bioactivity-guided isolation produced four active furanocoumarins, isoimperatorin (1), oxypeucedanin (2), oxypeucedanin hydrate (3) and imperatorin (4) from the n-BuOH fraction. Among them, compound 2 (followed by compounds 1, 4 and 3) showed a significant CPE inhibition effect, which was stronger than that of the positive control ribavirin, against both H1N1 and H9N2 with an EC50 (µM) of 5.98 ± 0.71 and 4.52 ± 0.39, respectively. Compound 2 inhibited the synthesis of NA and nucleoprotein (NP) in a dose-dependent manner. In the time course assays, the cytopathic effects of influenza A-infected MDCK cells were reduced by 80-90% when treated with compound 2 for 1 and 2 h after infection and declined drastically 3 h after infection. The level of viral NA and NP production was markedly reduced to less than 20% for both proteins in compound 2 (20 µM)-treated cells compared to untreated cells at 2 h after infection. In the molecular docking analysis, compound 2 showed a stronger binding affinity for the C-terminus of polymerase acidic protein (PAC; -36.28 kcal/mol) than the other two polymerase subunits. Compound 2 also exerted an antiapoptotic effect on virus infected cells and significantly inhibited the mRNA expression of caspase-3 and Bax. CONCLUSION: Our results suggest that compound 2 might exert anti-influenza A activity via the inhibition of the early phase of the viral replication cycle, not direct neutralization of surface proteins, such as hemagglutinin and NA, and abnormal apoptosis induced by virus infection. Taken together, these findings suggest that furanocoumarins predominant in A. dahurica play a pivotal role in its antiviral activity. These findings can also explain the reasons for the ethnopharmacological uses of this plant as an important ingredient in many antiviral prescriptions in traditional Chinese medicine (TCM).

Oleanane Triterpenoids from the Leaves of Gymnema inodorum and Their Insulin Mimetic Activities.

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.