Modulation of in Vitro SARS-CoV-2 Infection by Stephania tetrandra and Its Alkaloid Constituents.

Botanical natural products have been widely consumed for their purported usefulness against COVID-19. Here, six botanical species from multiple sources and 173 isolated natural product compounds were screened for blockade of wild-type (WT) SARS-CoV-2 infection in human 293T epithelial cells overexpressing ACE-2 and TMPRSS2 protease (293TAT). Antiviral activity was demonstrated by an extract from Stephania tetrandra. Extract fractionation, liquid chromatography-mass spectrometry (LC-MS), antiviral assays, and computational analyses revealed that the alkaloid fraction and purified alkaloids tetrandrine, fangchinoline, and cepharanthine inhibited WT SARS-CoV-2 infection. The alkaloids and alkaloid fraction also inhibited the delta variant of concern but not WT SARS-CoV-2 in VeroAT cells. Membrane permeability assays demonstrate that the alkaloids are biologically available, although fangchinoline showed lower permeability than tetrandrine. At high concentrations, the extract, alkaloid fractions, and pure alkaloids induced phospholipidosis in 293TAT cells and less so in VeroAT cells. Gene expression profiling during virus infection suggested that alkaloid fraction and tetrandrine displayed similar effects on cellular gene expression and pathways, while fangchinoline showed distinct effects on cells. Our study demonstrates a multifaceted approach to systematically investigate the diverse activities conferred by complex botanical mixtures, their cell-context specificity, and their pleiotropic effects on biological systems.

Liver Abnormalities after Elimination of HCV Infection: Persistent Epigenetic and Immunological Perturbations Post-Cure.

Chronic hepatitis C (CHC) is a major cause of hepatocellular carcinoma (HCC) worldwide. While directly acting antiviral (DAA) drugs are now able to cure virtually all hepatitis C virus (HCV) infections, even in subjects with advanced liver disease, what happens to the liver and progression of the disease after DAA-induced cure of viremia is only beginning to emerge. Several large-scale clinical studies in different patient populations have shown that patients with advanced liver disease maintain a risk for developing HCC even when the original instigator, the virus, is eliminated by DAAs. Here we review emerging studies derived from multiple, complementary experimental systems involving patient liver tissues, human liver cell cultures, human liver slice cultures, and animal models, showing that HCV infection induces epigenetic, signaling, and gene expression changes in the liver associated with altered hepatic innate immunity and liver cancer risk. Of critical importance is the fact that these virus-induced abnormalities persist after DAA cure of HCV. These nascent findings portend the discovery of pathways involved in post-HCV immunopathogenesis, which may be clinically actionable targets for more comprehensive care of DAA-cured individuals.

A validated UHPLC-tandem mass spectrometry method for quantitative analysis of flavonolignans in milk thistle (Silybum marianum) extracts.

Validated methods are needed for the analysis of natural product secondary metabolites. These methods are particularly important to translate in vitro observations to in vivo studies. Herein, a method is reported for the analysis of the key secondary metabolites, a series of flavonolignans and a flavonoid, from an extract prepared from the seeds of milk thistle [Silybum marianum (L.) Gaertn. (Asteraceae)]. This report represents the first UHPLC MS-MS method validated for quantitative analysis of these compounds. The method takes advantage of the excellent resolution achievable with UHPLC to provide a complete analysis in less than 7min. The method is validated using both UV and MS detectors, making it applicable in laboratories with different types of analytical instrumentation available. Lower limits of quantitation achieved with this method range from 0.0400µM to 0.160µM with UV and from 0.0800µM to 0.160µM with MS. The new method is employed to evaluate variability in constituent composition in various commercial S. marianum extracts, and to show that storage of the milk thistle compounds in DMSO leads to degradation.

Inhibition of HIV by Legalon-SIL is independent of its effect on cellular metabolism.

In this report, we further characterized the effects of silibinin (SbN), derived from milk thistle extract, and Legalon-SIL (SIL), a water-soluble derivative of SbN, on T cell metabolism and HIV infection. We assessed the effects of SbN and SIL on peripheral blood mononuclear cells (PBMC) and CEM-T4 cells in terms of cellular growth, ATP content, metabolism, and HIV infection. SIL and SbN caused a rapid and reversible (upon removal) decrease in cellular ATP levels, which was associated with suppression of mitochondrial respiration and glycolysis. SbN, but not SIL inhibited glucose uptake. Exposure of T cells to SIL (but not SbN or metabolic inhibitors) during virus adsorption blocked HIV infection. Thus, both SbN and SIL rapidly perturb T cell metabolism in vitro, which may account for its anti-inflammatory and anti-proliferative effects that arise with prolonged exposure of cells. However, the metabolic effects are not involved in SIL's unique ability to block HIV entry.

Silymarin for HCV infection.

Silymarin, an extract of milk thistle seeds, and silymarin-derived compounds have been considered hepatoprotective since the plant was first described in ancient times. Hepatoprotection is defined as several non-mutually exclusive biological activities including antiviral, antioxidant, anti-inflammatory and immunomodulatory functions. Despite clear evidence for silymarin-induced hepatoprotection in cell culture and animal models, evidence for beneficial effects in humans has been equivocal. This review will summarize the current state of knowledge on silymarin in the context of HCV infection. The information was collated from a recent workshop on silibinin in Germany.

Identification of hepatoprotective flavonolignans from silymarin.

Silymarin, also known as milk thistle extract, inhibits hepatitis C virus (HCV) infection and also displays antioxidant, anti-inflammatory, and immunomodulatory actions that contribute to its hepatoprotective effects. In the current study, we evaluated the hepatoprotective actions of the seven major flavonolignans and one flavonoid that comprise silymarin. Activities tested included inhibition of: HCV cell culture infection, NS5B polymerase activity, TNF-alpha-induced NF-kappaB transcription, virus-induced oxidative stress, and T-cell proliferation. All compounds were well tolerated by Huh7 human hepatoma cells up to 80 muM, except for isosilybin B, which was toxic to cells above 10 muM. Select compounds had stronger hepatoprotective functions than silymarin in all assays tested except in T cell proliferation. Pure compounds inhibited JFH-1 NS5B polymerase but only at concentrations above 300 muM. Silymarin suppressed TNF-alpha activation of NF-kappaB dependent transcription, which involved partial inhibition of IkappaB and RelA/p65 serine phosphorylation, and p50 and p65 nuclear translocation, without affecting binding of p50 and p65 to DNA. All compounds blocked JFH-1 virus-induced oxidative stress, including compounds that lacked antiviral activity. The most potent compounds across multiple assays were taxifolin, isosilybin A, silybin A, silybin B, and silibinin, a mixture of silybin A and silybin B. The data suggest that silymarin- and silymarin-derived compounds may influence HCV disease course in some patients. Studies where standardized silymarin is dosed to identify specific clinical endpoints are urgently needed.

Inhibition of T-cell inflammatory cytokines, hepatocyte NF-kappaB signaling, and HCV infection by standardized Silymarin.

BACKGROUND & AIMS: Chronic hepatitis C is a serious global medical problem necessitating effective treatment. Because standard of care with pegylated interferon plus ribavirin therapy is costly, has significant side effects, and fails to cure about half of all infections, many patients seek complementary and alternative medicine to improve their health, such as Silymarin, derived from milk thistle (Silybum marianum). Milk thistle's clinical benefits for chronic hepatitis C are unsettled due to variability in standardization of the herbal product. METHODS: In the current study, we focused on the anti-inflammatory and antiviral properties of a standardized Silymarin extract (MK-001). RESULTS: MK-001 inhibited expression of tumor necrosis factor-alpha in anti-CD3 stimulated human peripheral blood mononuclear cells and nuclear factor kappa B-dependent transcription in human hepatoma Huh7 cells. Moreover, MK-001 dose dependently inhibited infection of Huh7 and Huh7.5.1 cells by JFH-1 virus. MK-001 displayed both prophylactic and therapeutic effects against HCV infection, and when combined with interferon-alpha, inhibited HCV replication more than interferon-alpha alone. Commercial preparations of Silymarin also displayed antiviral activity, although the effects were not as potent as MK-001. Antiviral effects of the extract were attributable in part to induction of Stat1 phosphorylation, while interferon-independent mechanisms were suggested when the extract was biochemically fractionated by high-performance liquid chromatography. Silybin A, silybin B, and isosilybin A, isosilybin B elicited the strongest anti-NF-kappaB and anti-HCV actions. These effects were independent of MK-001-induced cytotoxicity. CONCLUSIONS: The data indicate that Silymarin exerts anti-inflammatory and antiviral effects, and suggest that complementary and alternative medicine-based approaches may assist in the management of patients with chronic hepatitis C.