Sangbaipi decoction exerted in vitro and in vivo anti-influenza effect through inhibiting viral proteins.

HEADINGS ETHNOPHARMACOLOGICAL RELEVANCE: Sangbaipi Decoction (SBPD) is an effective treatment for lung diseases caused by phlegm-heat obstruction according to Jingyue Quanshu, and soothes panting by purging the lung meridian. It is composed of anti-pyretic herbs (e.g., Scutellaria baicalensis Georgi and Coptis chinensis Franch.) and antitussive herbs (e.g., Cortex Mori and Armeniacae Semen Amarum). Therefore, we hypothesized that SBPD has therapeutic effects on lung injury caused by influenza virus. AIM OF THE STUDY: This study aimed to explore anti-influenza activity, active components, and mechanisms of SBPD. MATERIALS AND METHODS: The anti-influenza activities of SBPD were determined in 48 h drug-treated MDCK cell model using CPE and plaque reduction assays, and 24 h drug-treated A549 cells using qRT-PCR. The in vivo efficacy of SBPD (1.0 g/kg/day and 0.5 g/kg/day) was evaluated in PR8 infected BALB/c mice. The chemical component was assessed through HPLC-Q-TOF MS/MS analysis. Network pharmacology was built via TCMSP, GeneCards, DisgeNet, OMIM, DrugBank databases, and Cytoscape software. Additionally, TOA, HI and NAI assays were employed to investigate impact on the virus replication cycle with different concentrations of SBPD (2.5 mg/mL, 1.25 mg/mL, or 0.625 mg/mL). RESULTS: In MDCK infected with viruses A/PR/8/34, A/Hong Kong/1/68, or A/California/4/2009, the IC50 values of SBPD were 0.80 mg/mL, 1.20 mg/mL, and 1.25 mg/mL. In A549 cells, SBPD treatment reduced cytokine expression (e.g., TNF-α, IL-6, IL-1ß) (p < 0.05). In PR8 infected BALB/c mice, SBPD improved the survival rate of infected mice, reduced lung index (p < 0.05), protected lung tissue from pathological damage, and regulated cytokine overexpression (p < 0.05). 29 components of SBPD were identified in SBPD treated mouse serum including some phytochemicals targeting influenza proteins. HI and NAI assays suggested the potential antiviral mechanism of SBPD through inhibition of HA and NA. CONCLUSION: This study is the first to demonstrate the anti-influenza and the anti-inflammatory effects of SBPD in vitro and in vivo. Its major anti-influenza phytochemicals were explored and its inhibitory effects on HA and NA protein were proved. It provides more options for anti-influenza drug discovery.

Screening of growth inhibitors for epithelial-mesenchymal transition-induced cells by TGF-ß from plant-based sources identified the active compound hydroxychavicol from Piper bitle.

Epithelial-mesenchymal transition (EMT) has recently been associated with cancer invasion, metastasis, and resistance. In our previous study, we discovered nanaomycin K, a natural growth inhibitor for EMT-induced Madin Darby canine kidney (MDCK) cells, from the cultured broth of actinomycetes. However, the screening method was undeveloped, because the activity of nanaomycin K was discovered accidentally. In this study, we established a screening method by analyzing the characteristics of nanaomycin K in MDCK cells. Nanaomycin K showed the characteristic growth inhibitory activity on MDCK cells cultured under four conditions: medium containing dimethyl sulfoxide, SB431542, TGF-ß, and a mixture of SB431542 and TGF-ß. The activity was stronger in TGF-ß-treated cells than in DMSO-treated cells. In the mixture of SB431542 and TGF-ß-treated cells, the activity of nanaomycin K was suppressed. The anti-cancer agents, mitomycin C, cisplatin, and staurosporine, lacked the characteristics as that of nanaomycin K for these four treatment conditions. Since these four conditions distinguish between the effects of nanaomycin K and other anti-cancer agents in EMT-induced cells, the screening method was established. Among the 13,427 plant extracts tested, Piper betle leaf extract displayed growth inhibitory activity against EMT-induced cells. Through the purification of the extract via bio-guided fractionation, hydroxychavicol was isolated as an active compound. The cytotoxic activity of hydroxychavicol was stronger in EMT-induced MDCK cells than in control cells. However, its cytotoxic activity was suppressed in EMT-inhibited cells. Furthermore, hydroxychavicol exhibited same activity against SAS cells (human squamous cell carcinoma of the tongue). Thus, we have successfully established a screening method for growth inhibitors of EMT-induced cells and have discovered an inhibitor from plant-based sources.

Fu-Zheng-Xuan-Fei formula promotes macrophage polarization and Th17/Treg cell homeostasis against the influenza B virus (Victoria strain) infection.

ETHNOPHARMACOLOGICAL RELEVANCE: Fu-Zheng-Xuan-Fei formula (FF) is a prescription that has been clinically used through the basic theory of traditional Chinese medicine (TCM) for treating viral pneumonia. Although FF possesses a prominent clinical therapeutic effect, seldom pharmacological studies have been reported on its anti-influenza B virus (IBV) activity. AIM OF THE STUDY: Influenza is an acute infectious respiratory disease caused by the influenza virus, which has high annual morbidity and mortality worldwide. With a global decline in the COVID-19 control, the infection rate of influenza virus is gradually increasing. Therefore, it is of great importance to develop novel drugs for the effective treatment of influenza virus. Apart from conventional antiviral drugs, TCM has been widely used in the clinical treatment of influenza in China. Therefore, studying the antiviral mechanism of TCM can facilitate the scientific development of TCM. MATERIALS AND METHODS: Madin-Darby canine kidney cells (MDCK) and BALB/c mice were infected with IBV, and FF was added to evaluate the anti-IBV effects of FF both in vitro and in vivo by Western blotting, immunofluorescence, flow cytometry, and pathological assessment. RESULTS: It was found that FF exhibited anti-viral activity against IBV infection both in vivo and in vitro, while inducing macrophage activation and promoting M1 macrophage polarization. In addition, FF effectively regulated the signal transducer and activator of transcription (STAT) signaling pathway-mediated Th17/Treg balance to improve the lung tissue damage caused by IBV infection-induced inflammation. The findings provided the scientific basis for the antiviral mechanism of FF against IBV infection. CONCLUSIONS: This study shows that FF is a potentially effective antiviral drug against IBV infection.

Antiviral effect and mechanism of Phillyrin and its reformulated FS21 against influenza.

Background: Influenza virus causes significant morbidity and mortality with pandemic threat. Oleaceae Fructus Forsythiae is a medicinal herb. This study aimed to investigate antiviral effect of Phillyrin, a purified bioactive compound from this herb, and its reformulated preparation FS21 against influenza and its mechanism. Methods: Madin-Darby Canine Kidney (MDCK) cells were infected by one of six influenza viruses: five influenza A viruses (IAVs: three H1N1 and two H3N2) and one influenza B virus (IBV). Virus-induced cytopathic effects were observed and recorded under microscope. Viral replication and mRNA transcription were evaluated by quantitative polymerase chain reaction (qPCR) and protein expression by Western blot. Infectious virus production was assessed using TCID50 assay, and IC50 was calculated accordingly. Pretreatment and time-of-addition experiments with Phillyrin or FS21 added 1 h before or in early (0-3 h), mid (3-6 h), or late (6-9 h) stages of viral infection were performed to assess their antiviral effects. Mechanistic studies included hemagglutination and neuraminidase inhibition, viral binding and entry, endosomal acidification, and plasmid-based influenza RNA polymerase activity. Results: Phillyrin and FS21 had potent antiviral effects against all six IAV and IBV in a dose-dependent manner. Mechanistic studies showed that both suppressed influenza viral RNA polymerase with no effect on virus-mediated hemagglutination inhibition, viral binding or entry, endosomal acidification, or neuraminidase activity. Conclusions: Phillyrin and FS21 have broad and potent antiviral effects against influenza viruses with inhibition of viral RNA polymerase as the distinct antiviral mechanism.

Complex components of Shengmai formula interact with organic cation transporter 2 (OCT2) in MDCK cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Shengmai formula (SMF) is a well-known Chinese herbal compound preparation, which is utilized extensively for the treatment of myocardial ischemia, arrhythmia and other life-threatening conditions. Our previous researches have shown that some of the active ingredients in SMF can interact with organic anion transport polypeptide 1B1 (OATP1B1), breast cancer resistance protein (BCRP) and organic anion transporter 1 (OAT1), etc. Organic cation transporter 2 (OCT2) is a highly expressed uptake transporter in the kidney, and its interaction with the major active components in SMF remains unclear. AIM OF THE STUDY: We purposed to explore OCT2-mediated interactions and compatibility mechanisms of the main active compounds in SMF. MATERIALS AND METHODS: Fifteen active ingredients of SMF, including ginsenoside Rb1, Rd, Re, Rg1, Rf, Ro and Rc, methylophiopogonanone A and B, ophiopogonin D and D', schizandrin A and B, schizandrol A and B, were selected to investigate OCT2-mediated interactions in Madin-Darby cacine kidney (MDCK) cells stably expressing OCT2. RESULTS: Among the above 15 main active components, only ginsenosides Rd, Re and schizandrin B could significantly inhibit the uptake of 4-(4-(dimethylamino)styryl)-N-methyl pyridiniumiodide (ASP+), a classical substrate of OCT2. Ginsenoside Rb1 and methylophiopogonanone A can be transported by MDCK-OCT2 cells, and their uptake was significantly reduced when OCT2 inhibitor decynium-22 was added. Ginsenoside Rd could remarkably reduce the uptake of methylophiopogonanone A and ginsenoside Rb1 by OCT2, ginsenoside Re only decreased the uptake of ginsenoside Rb1, while schizandrin B had no effect on the uptake of both. CONCLUSIONS: OCT2 mediates the interaction of the major active components in SMF. Ginsenosides Rd, Re and schizandrin B are the potential inhibitors of OCT2, while ginsenosides Rb1 and methylophiopogonanone A are the potential substrates of OCT2. There is an OCT2-mediated compatibility mechanism among these active ingredients of SMF.

Anti-influenza properties of tiliroside isolated from Hibiscus mutabilis L.

ETHNOPHARMACOLOGICAL RELEVANCE: Fu Rong Ye (FRY), the leaf of Hibiscus mutabilis L., is a Chinese medicinal herb used to treat coughs and respiratory diseases. FRY is the major herbal component of the patent medicine Fupo Ganmao Granules for treating common cold. However, its anti-influenza active components and mechanism were not identified. AIM: Here, we aim to a) isolate the anti-influenza phytochemicals from FRY extract and b) explore its anti-flu mechanism. MATERIAL AND METHODS: Bioassay guided isolation was performed to get anti-influenza virus components. Influenza virus infected cells and mouse model were employed for efficacy evaluation. RESULTS: Using bioassay-guided isolation, the flavonoid tiliroside was obtained, which inhibited four IAV strains in MDCK cells with EC50 ranging from 3.87 to 27.61 µM by suppressing the viral ribonucleoprotein activity. Tiliroside also significantly downregulated the expression of cytokines/chemokines in A549 cells, and protected 50% of PR8-infected BALB/c mice from death and at 800 mg/kg/day, improved lung edema conditions. CONCLUSION: Tiliroside is effective for influenza virus infection treatment and promising for further drug development. This study is the first to demonstrate that tiliroside in FRY acts against influenza virus.

Effect of Oregon grape root extracts on P-glycoprotein mediated transport in in vitro cell lines.

Purpose: This study aims to investigate the potential of Oregon grape root extracts to modulate the activity of P-glycoprotein. Methods: We performed 3H-CsA or 3H-digoxin transport experiments in the absence or presence of two sources of Oregon grape root extracts (E1 and E2), berberine or berbamine in Caco-2 and MDCKII-MDR1 cells. In addition, real time quantitative polymerase chain reaction (RT-PCR) was performed in Caco-2 and LS-180 cells to investigate the mechanism of modulating P-glycoprotein. Results: Our results showed that in Caco-2 cells, Oregon grape root extracts (E1 and E2) (0.1-1 mg/mL) inhibited the efflux of CsA and digoxin in a dose-dependent manner. However, 0.05 mg/mL E1 significantly increased the absorption of digoxin. Ten µM berberine and 30 µM berbamine significantly reduced the efflux of CsA, while no measurable effect of berberine was observed with digoxin. In the MDCKII-MDR1 cells, 10 µM berberine and 30 µM berbamine inhibited the efflux of CsA and digoxin. Lastly, in real time RT-PCR study, Oregon grape root extract (0.1 mg/mL) up-regulated mRNA levels of human MDR1 in Caco-2 and LS-180 cells at 24 h. Conclusion: Our study showed that Oregon grape root extracts modulated P-glycoprotein, thereby may affect the bioavailability of drugs that are substrates of P-glycoprotein.

Metabolic reprogramming and alteration of the redox state in hyper-productive MDCK cells for influenza a virus production.

Influenza is a global public health issue leading to widespread morbidity and mortality with devastating economic loss annually. Madin-Darby Canine Kidney (MDCK) cell line has been a major cell line for influenza vaccine applications. Though many details of the host metabolic responses upon influenza A virus (IAV) infection have been documented, little is known about the metabolic reprogramming features of a hyper-productive host for IAV vaccine production. In this study, a MDCK cell clone H1 was shown to have a particular high productivity of 30 × 103 virions/cell. The glucose and amino acid metabolism of H1 were evaluated, indicating that the high producer had a particular metabolic reprogramming phenotype compared to its parental cell line (P): elevated glucose uptake, superior tricarboxylic acid cycle flux, moderate amino acid consumption, and better regulation of reactive oxygen species. Combined with the stronger mitochondrial function and mild antiviral and inflammatory responses characterized previously, our results indicated that the high producer had a sufficient intracellular energy supply, and balanced substrate distribution for IAV and host protein synthesis as well as the intracellular redox status. Understanding of these metabolic alterations paves the way for the rational cell line development and reasonable process optimization for high-yield influenza vaccine production.

Urolithin M5 from the Leaves of Canarium album (Lour.) DC. Inhibits Influenza Virus by Targeting Neuraminidase.

Ganlanye (GLY), the leaf of Canarium album (Lour.) DC., is a traditional Chinese medicinal herb for warm disease treatment. We found that its aqueous extract could inhibit the influenza A virus. To find and characterize anti-influenza virus phytochemicals from GLY, we performed (1) bioassay-guided isolation, (2) a cell and animal assay, and (3) a mechanism study. Bioassay-guided isolation was used to identify the effective components. Influenza virus-infected MDCK cell and BALB/c mouse models were employed to evaluate the anti-influenza virus activities. A MUNANA assay was performed to find the NA inhibitory effect. As a result, urolithin M5 was obtained from the crude extract of GLY. It inhibited influenza virus activities in vitro and in vivo by suppressing the viral NA activity. In the MDCK cell model, urolithin M5 could inhibit an oseltamivir-resistant strain. In a PR8-infected mouse model, 200 mg/kg/d urolithin M5 protected 50% of mice from death and improved lung edema conditions. GLY was recorded as a major traditional herb for warm disease treatment. Our study identified GLY as a potent anti-influenza herb and showed urolithin M5 as the active component. We first report the in vivo activity of urolithin M5 and support the anti-influenza application of GLY.

Screening of Antiviral Components of Yinhuapinggan Granule and Protective Effects of Yinhuapinggan Granule on MDCK Cells with Influenza A/H1N1 Virus.

BACKGROUND: Traditional Chinese medicine Yinhuapinggan granule (YHPG) has been used for treating upper respiratory tract infection like influenza, cough, and viral pneumonia. However, its active ingredients that really exert the main efficacy have not been well elucidated. This study is aimed at screening its antiviral components and investigating the potential therapeutic mechanisms of YHPG against the influenza A/PR8/34 (H1N1) virus in Madin Darby canine kidney (MDCK). METHODS: MDCK cells were infected with the influenza virus and then treated with ribavirin, YHPG, and main active ingredients in YHPG. Based on the maximum nontoxic concentration (TC0), half-maximal toxic concentration (TC50), half-maximal inhibitory concentration (IC50), and therapeutic index (TI), interferon-ß (IFN-ß) and interleukin-6 (IL-6) levels were measured using enzyme-linked immunosorbent assay (ELISA), and the gene expression of TLR7, MyD88, tumor necrosis factor receptor-associated factor 6 (TRAF6), c-Jun amino terminal kinase (JNK), p38 mitogen-activated protein kinase (p38 MAPK), and p65 nuclear transcription factor-kappa B (p65 NF-κB) was quantified using reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: The results indicated that the components of YHPG, such as ephedrine hydrochloride, pseudoephedrine hydrochloride, chlorogenic acid, and emodin, had significant antiviral effects. High and medium doses of YHPG effectively reduced the cytopathic effect (CPE) and significantly decreased IFN-ß and IL-6 levels in the supernatant. Simultaneously, the transcript levels of TLR7, MyD88, TRAF6, JNK, p38 MAPK, and p65 NF-κB decreased in infected MDCK cells. Moreover, a certain dose-dependent relationship among different groups of YHPG was observed. CONCLUSIONS: These results indicated that YHPG and the components of YHPG had a significant inhibitory function on the proliferation of the H1N1 virus. The mechanism might be associated with suppressing the activation of the TLR7/MyD88 signaling pathway, a decrease in the mRNA expression of key target genes, and inhibition of IFN-ß and IL-6 secretion.

Anti-Influenza Activity of Medicinal Material Extracts from Qinghai-Tibet Plateau.

To discover sources for novel anti-influenza drugs, we evaluated the antiviral potential of nine extracts from eight medicinal plants and one mushroom (Avena sativa L., Hordeum vulgare Linn. var. nudum Hook. f., Hippophae rhamnoides Linn., Lycium ruthenicum Murr., Nitraria tangutorum Bobr., Nitraria tangutorum Bobr. by-products, Potentilla anserina L., Cladina rangiferina (L.) Nyl., and Armillaria luteo-virens) from the Qinghai-Tibetan plateau against the influenza A/H3N2 virus. Concentrations lower than 125 µg/mL of all extracts demonstrated no significant toxicity in MDCK cells. During screening, seven extracts (A. sativa, H. vulgare, H. rhamnoides, L. ruthenicum, N. tangutorum, C. rangiferina, and A. luteo-virens) exhibited antiviral activity, especially the water-soluble polysaccharide from the fruit body of the mushroom A. luteo-virens. These extracts significantly reduced the infectivity of the human influenza A/H3N2 virus in vitro when used at concentrations of 15.6-125 µg/mL. Two extracts (N. tangutorum by-products and P. anserina) had no A/H3N2 virus inhibitory activity. Notably, the extract obtained from the fruits of N. tangutorum and N. tangutorum by-products exhibited different anti-influenza effects. The results suggest that extracts of A. sativa, H. vulgare, H. rhamnoides, L. ruthenicum, N. tangutorum, C. rangiferina, and A. luteo-virens contain substances with antiviral activity, and may be promising sources of new antiviral drugs.

Application of a high-resolution in vitro human MDR1-MDCK assay and in vivo studies in preclinical species to improve prediction of CNS drug penetration.

P-glycoprotein (P-gp, MDR1) is expressed at the blood-brain barrier (BBB) and restricts penetration of its substrates into the central nervous system (CNS). In vitro MDR1 assays are frequently used to predict the in vivo relevance of MDR1-mediated efflux at the BBB. It has been well established that drug candidates with high MDR1 efflux ratios (ERs) display poor CNS penetration. Following a comparison of MDR1 transporter function between the MDR1-MDCKI cell line from National Institutes of Health (NIH) and our internal MDR1-MDCKII cell line, the former was found to provide better predictions of in vivo brain penetration than our in-house MDR1-MDCKII cell line. In particular, the NIH MDR1 assay has an improved sensitivity to differentiate the compounds with ERs of <3 in our internal cell line and is able to reduce the risk of false negatives. A better correlation between NIH MDR1 ERs and brain penetration in rat and non-human primate (NHP) was demonstrated. Additionally, a comparison of brain penetration time course of MDR1 substrates and an MDR1 non-substrate in NHP demonstrated that MDR1 interaction can delay the time to equilibrium of drug concentration in the brain with plasma. It is recommended to select highly permeable compounds without MDR1 interaction for rapid brain penetration to produce the maximal pharmacological effect in the CNS with a quicker onset.

Facial Synthesis and Bioevaluation of Well-Defined OEGylated Betulinic Acid-Cyclodextrin Conjugates for Inhibition of Influenza Infection.

Betulinic acid (BA) and its derivatives exhibit a variety of biological activities, especially their anti-HIV-1 activity, but generally have only modest inhibitory potency against influenza virus. The entry of influenza virus into host cells can be competitively inhibited by multivalent derivatives targeting hemagglutinin. In this study, a series of hexa-, hepta- and octavalent BA derivatives based on α-, ß- and γ-cyclodextrin scaffolds, respectively, with varying lengths of flexible oligo(ethylene glycol) linkers was designed and synthesized using a microwave-assisted copper-catalyzed 1,3-dipolar cycloaddition reaction. The generated BA-cyclodextrin conjugates were tested for their in vitro activity against influenza A/WSN/33 (H1N1) virus and cytotoxicity. Among the tested compounds, 58, 80 and 82 showed slight cytotoxicity to Madin-Darby canine kidney cells with viabilities ranging from 64 to 68% at a high concentration of 100 µM. Four conjugates 51 and 69-71 showed significant inhibitory effects on influenza infection with half maximal inhibitory concentration values of 5.20, 9.82, 7.48 and 7.59 µM, respectively. The structure-activity relationships of multivalent BA-cyclodextrin conjugates were discussed, highlighting that multivalent BA derivatives may be potential antiviral agents against influenza infection.

Intestinal absorption mechanism of rotundic acid: Involvement of P-gp and OATP2B1.

ETHNOPHARMACOLOGICAL RELEVANCE: Ilicis Rotundae Cortex (IRC), the dried barks of Ilex rotunda Thunb. (Aquifoliaceae), has been used for the prevention or treatment of colds, tonsillitis, dysentery, and gastrointestinal diseases in folk medicine due to its antibacterial and anti-inflammatory effects. However, there is no report about the intestinal absorption of major compounds that support traditional usage. AIM OF STUDY: Considering the potential of rotundic acid (RA) - major biologically active pentacyclic triterpenes found in the IRC, this study was purposed to uncover the oral absorption mechanism of RA using in situ single-pass intestinal perfusion (SPIP) model, in vitro cell models (Caco-2, MDCKII-WT, MDCKII-MDR1, MDCKII-BCRP, and HEK293-OATP2B1 cells) and in vivo pharmacokinetics studies in rats. MATERIALS AND METHODS: The molecular properties (solubility, lipophilicity, and chemical stability) and the effects of principal parameters (time, compound concentrations, pH, paracellular pathway, and the different intestinal segments) were analyzed by liquid chromatography-tandem mass spectrometry. The susceptibility of RA to various inhibitors, such as P-gp inhibitor verapamil, BCRP inhibitor Ko143, OATP 2B1 inhibitor rifampicin, and absorption enhancer EGTA were assessed. RESULTS: RA was a compound with low water solubility (12.89 µg/mL) and strong lipophilicity (LogP = 4.1). RA was considered stable in all media during the SPIP and transport studies. The SPIP and cell experiments showed RA was moderate absorbed in the intestines and exhibited time, concentration, pH, and segment-dependent permeability. In addition, results from the cell model, in situ SPIP model as well as the in vivo pharmacokinetics studies consistently showed that verapamil, rifampicin, and EGTA might have significant effect on the intestinal absorption of RA. CONCLUSION: The mechanisms of intestinal absorption of RA might involve multiple transport pathways, including passive diffusion, the participation of efflux (i.e., P-gp) and influx (i.e., OATP2B1) transporters, and paracellular pathways.

Effects of medicagenic acid metabolites, originating from biotransformation of an Herniaria hirsuta extract, on calcium oxalate crystallization in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: Herniaria hirsuta is traditionally used in Moroccan folk medicine for treatment of urinary stones and as a diuretic. It is rich in saponins, which are known to be deglycosylated in the colon, whereafter aglycones such as medicagenic acid are absorbed and further metabolized in the liver. AIM OF THE STUDY: A sample of hepatic metabolites of medicagenic acid, with medicagenic acid glucuronide as the most abundant one, was evaluated for in vitro activity against urinary stones. A crystallization assay and a crystal-cell interaction assay were used to evaluate in vitro activity of hepatic metabolites of medicagenic acid on CaC2O4 (calciumoxalate) crystals, present in the majority of urinary stones. MATERIALS AND METHODS: In the crystallization assay the effects on nucleation of Ca2+ and C2O42- and aggregation of the CaC2O4 crystals are studied. In the crystal-cell interaction assay crystal retention is investigated by determining the amount of Ca2+ bound to injured monolayers of MDCK I cells. RESULTS: Results of the crystallization assay showed a tentative effect on crystal aggregation. The crystal-cell interaction assay showed a significant inhibition of crystal binding, which may reduce crystal retention in the urinary tract. CONCLUSIONS: As both formation of crystals by inhibiting aggregation and retention of crystals is affected, the beneficial effect of H. hirsuta against urinary stones may at least in part be attributed to medicagenic acid metabolites, indicating that saponins containing medicagenic acid may act as prodrugs.

New anti-influenza A viral norsesquiterpenoids isolated from feces-residing Streptomyces sp.

Three novel norsesquiterpenoids, (2R,4S,8aR)-8,8a,1,2,3,4-hexahydro-2-hydroxy-4,8a-dimethyl-2(2H)-naphthalenone (1), (1S,3S,4S,4aS,8aR)-4,8a-dimethyloctahydronaphthalene-1,3,4a(3H)-triol(2), (4S,4aS,8aS)-octahydro-4a-hydroxy-4, 8a-dimethyl-1(2H)-naphthalenone (3), as well as six other known analogues (4-9), were isolated from the culture broth of Streptomyces sp. XM17, an actinobacterial strain inhabiting the fresh feces of the giant panda Ailuropoda melanoleuca. The chemical structures of 1-3 were elucidated comprehensively by NMR spectroscopic and MS analyses, furthermore, the stereochemical configurations were resolved by NOESY experiments, along with ECD spectral and single-crystal X-ray crystallographic analyses. These compounds were then tested for their antiviral activities using the "pretreatment of virus" approach, which showed that most of these compounds were potent in inhibiting the entry of influenza A virus, with IC50 values ranging from 5 to 49 nM and selectivity indices all above 500.

Epigallocatechin-3-Gallate as a Novel Vaccine Adjuvant.

Vaccine adjuvants from natural resources have been utilized for enhancing vaccine efficacy against infectious diseases. This study examined the potential use of catechins, polyphenolic materials derived from green tea, as adjuvants for subunit and inactivated vaccines. Previously, catechins have been documented to have irreversible virucidal function, with the possible applicability in the inactivated viral vaccine platform. In a mouse model, the coadministration of epigallocatechin-3-gallate (EGCG) with influenza hemagglutinin (HA) antigens induced high levels of neutralizing antibodies, comparable to that induced by alum, providing complete protection against the lethal challenge. Adjuvant effects were observed for all types of HA antigens, including recombinant full-length HA and HA1 globular domain, and egg-derived inactivated split influenza vaccines. The combination of alum and EGCG further increased neutralizing (NT) antibody titers with the corresponding hemagglutination inhibition (HI) titers, demonstrating a dose-sparing effect. Remarkably, EGCG induced immunoglobulin isotype switching from IgG1 to IgG2a (approximately >64-700 fold increase), exerting a more balanced TH1/TH2 response compared to alum. The upregulation of IgG2a correlated with significant enhancement of antibody-dependent cellular cytotoxicity (ADCC) function (approximately 14 fold increase), providing a potent effector-mediated protection in addition to NT and HI. As the first report on a novel class of vaccine adjuvants with built-in virucidal activities, the results of this study will help improve the efficacy and safety of vaccines for pandemic preparedness.

Magnolol and Honokiol Inhibited the Function and Expression of BCRP with Mechanism Exploration.

Breast cancer resistance protein (BCRP), one of the ATP-binding cassette (ABC) transporters, was associated with the multidrug resistance (MDR) of chemotherapy. Magnolol (MN) and honokiol (HK) are major bioactive polyphenols of Magnolia officinalis. This study investigated the effects of MN and HK on the function and expression of BCRP for the purpose of developing BCRP inhibitor to overcome MDR. Cell lines including MDCKII-BCRP and MDCKII-WT were used for evaluating the function and expression of BCRP. The results showed that MN (100-12.5 µM) and HK (100-12.5 µM) significantly decreased the function of BCRP by 80~12% and 67~14%, respectively. In addition, MN and HK were verified as substrates of BCRP. Furthermore, MN and HK reduced the protein expression of BCRP, and inhibited the phosphorylation of epidermal growth factor receptor (EGFR) and phosphatidylinositol 3-kinase (PI3K). In conclusion, both MN and HK decreased the function and expression of BCRP via EGFR/PI3K signaling pathway. Therefore, both compounds were promising candidates for reversing the MDR of chemotherapy.

Erianin: A Direct NLRP3 Inhibitor With Remarkable Anti-Inflammatory Activity.

Erianin (Eri) is the extract of Dendrobium chrysotoxum Lindl. The NLRP3 inflammasome is a multiprotein complex that plays key roles in a wide variety of chronic inflammation-driven human diseases. Nevertheless, little is known about the protection of Eri against NLRP3 inflammasome-related diseases. In this study, we demonstrated that Eri inhibited NLRP3 inflammasome activation in vitro and in vivo. Mechanistically, Eri directly interacted with NLRP3, leading to inhibition of NLRP3 inflammasome assembly. Eri associated with the Walker A motif in the NACHT domain and suppressed NLRP3 ATPase activity. In mouse models, Eri had therapeutic effects on peritonitis, gouty arthritis and type 2 diabetes, via NLRP3. More importantly, Eri was active ex vivo for synovial fluid cells and monocytes from patients with IAV infection and gout. Eri may serve as a potential novel therapeutic compound against NLRP3-driven diseases.

Antiviral Activity and Underlying Action Mechanism of Euglena Extract against Influenza Virus.

It is difficult to match annual vaccines against the exact influenza strain that is spreading in any given flu season. Owing to the emergence of drug-resistant viral strains, new approaches for treating influenza are needed. Euglena gracilis (hereinafter Euglena), microalga, used as functional foods and supplements, have been shown to alleviate symptoms of influenza virus infection in mice. However, the mechanism underlying the inhibitory action of microalgae against the influenza virus is unknown. Here, we aimed to study the antiviral activity of Euglena extract against the influenza virus and the underlying action mechanism using Madin-Darby canine kidney (MDCK) cells. Euglena extract strongly inhibited infection by all influenza virus strains examined, including those resistant to the anti-influenza drugs oseltamivir and amantadine. A time-of-addition assay revealed that Euglena extract did not affect the cycle of virus replication, and cell pretreatment or prolonged treatment of infected cells reduced the virus titer. Thus, Euglena extract may activate the host cell defense mechanisms, rather than directly acting on the influenza virus. Moreover, various minerals, mainly zinc, in Euglena extract were found to be involved in the antiviral activity of the extract. In conclusion, Euglena extract could be a potent agent for preventing and treating influenza.