Veratramine inhibits porcine epidemic diarrhea virus entry through macropinocytosis by suppressing PI3K/Akt pathway.

Porcine epidemic diarrhea (PED) is a contagious intestinal disease caused by α-coronavirus porcine epidemic diarrhea virus (PEDV). At present, no effective vaccine is available to prevent the disease. Therefore, research for novel antivirals is important. This study aimed to identify the antiviral mechanism of Veratramine (VAM), which actively inhibits PEDV replication with a 50 % inhibitory concentration (IC50) of ∼5 µM. Upon VAM treatment, both PEDV-nucleocapsid (N) protein level and virus titer decreased significantly. The time-of-addition assay results showed that VAM could inhibit PEDV replication by blocking viral entry. Importantly, VAM could inhibit PEDV-induced phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) activity and further suppress micropinocytosis, which is required for PEDV entry. In addition, PI3K inhibitor LY294002 showed anti-PEDV activity by blocking viral entry as well. Taken together, VAM possessed anti-PEDV properties against the entry stage of PEDV by inhibiting the macropinocytosis pathway by suppressing the PI3K/Akt pathway. VAM could be considered as a lead compound for the development of anti-PEDV drugs and may be used during the viral entry stage of PEDV infection.

Antihypertensive activity of different components of Veratrum alkaloids through metabonomic data analysis.

BACKGROUND: Hypertension is a serious global public health issue. Blood pressure (BP) is still not effectively controlled in about 20 - 30% of hypertensive patients. Therefore, it is imperative to develop new treatments for hypertension. Veratrum alkaloids were once used for the clinical treatment of hypertension, the mechanism of which is still unclear. It was gradually phased out due to adverse reactions. PURPOSE: This study aimed to investigate the short-term and long-term hypotensive profiles of different components of Veratrum alkaloids in spontaneously hypertensive rats (SHRs) to unveil their mechanisms of action. RESULTS: Total Veratrum alkaloid (V), component A (A), and veratramine (M) quickly decreased BP within 30 min of treatment, reduced renal and cardiovascular damage, and improved relevant biochemical indicators (nitric oxide [NO], endothelin-1 [ET-1], angiotensin II [Ang II)], noradrenaline [NE], etc) in SHRs to delay stroke occurrence. Thereinto, A exhibited excellent protective effects in cardiovascular disease. The metabolomic profiles of SHRs treated with V, A, and M were significantly different from those of SHRs treated with vehicle. Thirteen metabolites were identified as potential pharmacodynamic biomarkers. Through Kyoto Encyclopedia of Genes and Genomes analysis, V, A, and M-induced hypotension was mainly related to alterations in nicotinate and nicotinamide metabolism, GABAergic synapses, linoleic acid metabolism, ketone body synthesis and degradation, arginine and proline metabolism, and urea cycle, of which nicotinate and nicotinamide metabolism was the key metabolic pathway to relieve hypertension. CONCLUSION: This work shows that A is an effective and promising antihypertensive agent for hypertension treatment to reduce BP and hypertensive target organ damage, which is mainly mediated through modulating nicotinate and nicotinamide metabolism, RAS, and NO-ET homeostasis.

Veratramine Inhibits the Cell Cycle Progression, Migration, and Invasion via ATM/ATR Pathway in Androgen-Independent Prostate Cancer.

Prostate cancer (PC) is the second leading cause of cancer-related death among men. Treatment of PC becomes difficult after progression because PC that used to be androgen-dependent becomes androgen-independent prostate cancer (AIPC). Veratramine, an alkaloid extracted from the root of the Veratrum genus, has recently been reported to have anticancer effects that work against various cancers; however, its anticancer effects and the underlying mechanism of action in PC remain unknown. We investigated the anticancer effects of veratramine on AIPC using PC3 and DU145 cell lines, as well as a xenograft mouse model. The antitumor effects of veratramine were evaluated using the CCK-8, anchorage-independent colony formation, trans-well, wound healing assays, and flow cytometry in AIPC cell lines. Microarray and proteomics analyses were performed to investigate the differentially expressed genes and proteins induced by veratramine in AIPC cells. A xenograft mouse model was used to confirm the therapeutic response and in vivo efficacy of veratramine. Veratramine dose dependently reduced the proliferation of cancer cells both in vitro and in vivo. Moreover, veratramine treatment effectively suppressed the migration and invasion of PC cells. The immunoblot analysis revealed that veratramine significantly downregulated Cdk4/6 and cyclin D1 via the ATM/ATR and Akt pathways, both of which induce a DNA damage response that eventually leads to G1 phase arrest. In this study, we discovered that veratramine exerted antitumor effects on AIPC cells. We demonstrated that veratramine significantly inhibited the proliferation of cancer cells via G0/G1 phase arrest induced by the ATM/ATR and Akt pathways. These results suggest that veratramine is a promising natural therapeutic agent for AIPC.

The Effect of Botanical Pesticides Azadirachtin, Celangulin, and Veratramine Exposure on an Invertebrate Species Solenopsis invicta (Hymenoptera: Formicidae).

The injudicious and excessive use of synthetic pesticides has deleterious effects on humans, ecosystems, and biodiversity. As an alternative to traditional crop-protection methods, botanical pesticides are gaining importance. In this research endeavor, we examined the contact toxicity, knockdown time, lethal time, and toxicity horizontal transmission of three natural pesticides from plants (azadirachtin, celangulin, and veratramine) on red imported fire ants (RIFA; Solenopsis invicta). Our research findings indicated that azadirachtin and celangulin exhibited relatively high toxicity, with median lethal dose (LD50) values of 0.200 and 0.046 ng/ant, respectively, whereas veratramine exhibited an LD50 value of 544.610 ng/ant for large workers of S. invicta at 24 h post-treatment. Upon treatment with 0.125 mg/L, the (median lethal time) LT50 values of azadirachtin and celangulin were determined to be 60.410 and 9.905 h, respectively. For veratramine, an LT50 value of 46.967 h was achieved after being tested with 200 mg/L. Remarkably, azadirachtin and celangulin were found to exhibit high horizontal transfer among RIFA, with high secondary mortality (100%) and tertiary mortalities (>61%) after 48 h of treatment with 250 mg/L, as well as with their dust formulations for 72 h. However, veratramine did not exhibit significant toxicity or horizontal transfer effects on RIFA, even at high concentrations. These findings suggest that azadirachtin and celangulin are likely to have a highly prominent potential in the management of S. invicta.

Anticancer effects of veratramine via the phosphatidylinositol-3-kinase/serine-threonine kinase/mechanistic target of rapamycin and its downstream signaling pathways in human glioblastoma cell lines.

AIMS: Antitumor effects of veratramine in prostate and liver cancers has been investigated, but it is still unclear whether veratramine can be used as an effective therapeutic agent for glioma. The aim of this study was to evaluate the potential pharmacological mechanism of veratramine in glioma. MAIN METHODS: Using four types of human glioblastoma cell lines, including A172, HS-683, T98G, and U-373-MG the dose-dependent antitumor effect of veratramine was evaluated. The cytotoxicity and cell proliferation were examined by CCK-8, and cell proliferation was further confirmed by anchorage-independent colony formation assay. The cell cycle distribution and apoptotic rate was assessed by flow cytometry, and apoptosis was further evaluated by apoptosis assay. The migration and invasiveness capacity were analyzed by using transwell. Protein and mRNA levels of related factors were determined by western blotting and RT-qPCR, respectively. KEY FINDINGS: Veratramine markedly induced apoptosis, suppressed the cell proliferation via the cell cycle G0/G1 phase arrest, and reduced the capacity for the migration and invasion in human glioblastoma multiforme cell lines. Moreover, veratramine was sufficient to affect the phosphatidylinositol-3-kinase/serine-threonine kinase/mechanistic target of rapamycin signaling pathway and its downstream Mdm2/p53/p21 pathway in human glioblastoma cell lines. SIGNIFICANCE: Antitumor effects of veratramine in suppression of glioma progression was mediated by the regulation of PI3K/Akt/mTOR and Mdm2/p53/p21 signaling pathway.

Veratramine suppresses human HepG2 liver cancer cell growth in vitro and in vivo by inducing autophagic cell death.

Liver cancer is the second leading cause of cancer­related deaths. Traditional therapeutic strategies, such as chemotherapy, targeted therapy and interventional therapy, are inefficient and are accompanied by severe side effects for patients with advanced liver cancer. Therefore, it is crucial to develop a safer more effective drug to treat liver cancer. Veratramine, a known natural steroidal alkaloid derived from plants of the lily family, exerts anticancer activity in vitro. However, the underlying mechanism and whether it has an antitumor effect in vivo remain unknown. In the present study, the data revealed that veratramine significantly inhibited HepG2 cell proliferation, migration and invasion in vitro. Moreover, it was revealed that veratramine induced autophagy­mediated apoptosis by inhibiting the PI3K/Akt/mTOR signaling pathway, which partly explained the underlying mechanism behind its antitumor activity. Notably, the results of in vivo experiments also revealed that veratramine treatment (2 mg/kg, 3 times a week for 4 weeks) significantly inhibited subcutaneous tumor growth of liver cancer cells, with a low systemic toxicity. Collectively, the results of the present study indicated that veratramine efficiently suppressed liver cancer HepG2 cell growth in vitro and in vivo by blocking the PI3K/Akt/mTOR signaling pathway to induce autophagic cell death. Veratramine could be a potential therapeutic agent for the treatment of liver cancer.

Seven new veratramine-type alkaloids with potent analgesic effect from Veratrum taliense.

ETHNOPHARMACOLOGICAL RELEVANCE: Veratrum taliense is traditionally used TCMs in Yunnan province of China for pain and inflammation. Previous research and clinical applications have shown that V. taliense had significant analgesic activity. Jevine-type alkaloids were shown to be one of the anti-inflammatory and analgesic agents from V. taliense. However, other types of compounds from V. taliense related to its traditional use remains unknown. AIM OF THE STUDY: To identify veratramine-type steroidal alkaloids with analgesic effects from the roots and rhizomes of V. taliense. MATERIALS AND METHODS: Compounds were isolated from the roots and rhizomes of V. taliense by chromatographic separation. Their structures were elucidated based on UV, IR, NMR and MS spectra data. Analgesic activity was assessed with acetic acid-induced writhing in mice model. RESULTS: Seven new veratramine-type alkaloids were isolated from the roots and rhizomes of V. taliense. They all exhibited significant analgesic activity, of which alkaloids 1 and 4 were more potent antalgic than the well-known analgesic drug, pethidine. CONCLUSIONS: The veratramine-type alkaloids from V. taliense may serve as new leads for the discovery of analgesic drugs.

Pharmacokinetics and metabolism study of veratramine in mice after oral administration using LC-MS/MS.

A simple and sensitive high-performance liquid chromatography coupled with hybrid triple quadrupole-linear ion trap mass spectrometry (Q-trap-MS) method was developed and validated for the determination of veratramine, the major bioactive and neurotoxic component in Veratrum nigrum L. Veratramine and the internal standard (IS) were separated with a Waters Symmetry C18 column and eluted with a gradient mobile phase system containing acetonitrile and 0.1% aqueous formic acid. The analysis was performed by using positive electrospray ionization mode with multiple reaction monitoring (MRM). Transition ions of m/z 410.2 → 295.2 for veratramine and m/z 426.1 → 113.8 for the IS were monitored. The method was validated with a good linearity in the range of 1-1000 ng/mL and lower limit of quantification of 1 ng/mL. The precision (CV) of intra- and inter-day ranged from 3.92 to 7.29%, while the accuracy (bias) intra- and inter-day were between -4.78 and 1.65%. The recovery, stability and matrix effect were within the acceptable ranges. Five metabolites of veratramine, including four hydroxylated and one sulfated metabolites, were tentatively identified using predictive MRM-information dependent acquisition-enhanced product ion mode (predictive MRM-IDA-EPI). The developed method was successfully applied to the pharmacokinetic and metabolic study of veratramine in mice after oral administration of veratramine. Copyright © 2016 John Wiley & Sons, Ltd.

Gender-Dependent Pharmacokinetics of Veratramine in Rats: In Vivo and In Vitro Evidence.

Veratramine, a major alkaloid from Veratrum nigrum L., has distinct anti-tumor and anti-hypertension effects. Our previous study indicated that veratramine had severe toxicity toward male rats. In order to elucidate the underling mechanism, in vivo pharmacokinetic experiments and in vitro mechanistic studies have been conducted. Veratramine was administrated to male and female rats intravenously via the jugular vein at a dose of 50 µg/kg or orally via gavage at 20 mg/kg. As a result, significant pharmacokinetic differences were observed between male and female rats after oral administration with much lower concentrations of veratramine and 7-hydroxyl-veratramine and higher concentrations of veratramine-3-O-sulfate found in the plasma and urine of female rats. The absolute bioavailability of veratramine was 0.9% in female rats and 22.5% in male rats. Further experiments of veratramine on Caco-2 cell monolayer model and in vitro incubation with GI content or rat intestinal subcellular fractions demonstrated that its efficient passive diffusion mediated absorption with minimal intestinal metabolism, suggesting no gender-related difference during its absorption process. When veratramine was incubated with male or female rat liver microsomes/cytosols, significant male-predominant formation of 7-hydroxyl-veratramine and female-predominant formation of veratramine-3-O-sulfate were observed. In conclusion, the significant gender-dependent hepatic metabolism of veratramine could be the major contributor to its gender-dependent pharmacokinetics.

Identification and characterization of in vitro and in vivo metabolites of steroidal alkaloid veratramine.

Veratramine, a steroidal alkaloid originating from Veratrum nigrum L., has demonstrated distinct anti-tumor and anti-hypertension effects, however, its metabolism has rarely been explored. The objective of the current study was to provide a comprehensive investigation of its metabolic pathways. The in vitro metabolic profiles of veratramine were evaluated by incubating it with liver microsomes and cytosols. The in vivo metabolic profiles in plasma, bile, urine and feces were monitored by UPLC-MS/MS after oral (20 mg/kg) and i.v. (50 µg/kg) administration in rats. Meanwhile, related P450s inhibitors and recombinant P450s and SULTs were used to identify the isozymes responsible for its metabolism. Eleven metabolites of veratramine, including seven hydroxylated, two sulfated and two glucuronidated metabolites, were characterized. Unlike most alkaloids, the major reactive sites of veratramine were on ring A and B instead of on the amine moiety. CYP2D6 was the major isozyme mediating hydroxylation, and substrate inhibition was observed with a Vmax , Ki and Clint of 2.05 ± 0.53 nmol/min/mg, 33.08 ± 10.13 µ m and 13.58 ± 1.27 µL/min/mg. SULT2A1, with Km , Vmax and Clint values of 19.37 ± 0.87 µ m, 1.51 ± 0.02 nmol/min/mg and 78.19 ± 8.57 µL/min/mg, was identified as the major isozyme contributing to its sulfation. In conclusion, CYP2D6 and SULT2A1 mediating hydroxylation and sulfation were identified as the major biotransformation for veratramine.