Anticoronavirus activity of rhizome of Dryopteris crassirhizoma via multistage targeting of virus entry and viral proteases, Mpro and PLpro.

ETHNOPHARMACOLOGICAL RELEVANCE: The rhizome of Dryopteris crassirhizoma Nakai (Dryopteridaceae, RDC), a traditional East Asian herbal medicine, possesses a broad spectrum of medicinal properties, including anti-inflammatory, anticancer, antibacterial, and antiviral activities. AIM OF THE STUDY: This study investigates the 30% ethanolic extract of RDC's antiviral potential against human coronavirus OC43 (HCoV-OC43), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and its variants infections. MATERIALS AND METHODS: A 30% ethanolic extract of RDC or its components, filixic acid ABA (PubChem CID: 15081408) and dryocrassin ABBA (PubChem CID: 3082025) were treated with Human Coronavirus infection (HCoV-OC43, SARS-CoV-2 and its variants). The base peak chromatogram of RDC was evaluated using UPLC-Q/TOF Mass to identify the RDC, and the quantitative analysis of RDC compounds was performed using LC-MS/MS. A cytopathic effect (CPE) reduction assay, Western blot, immunofluorescence staining of viral protein expression, and qRT-PCR were performed to quantify the viral RNA copy numbers and determine the antiviral activity. The time-of-addition assay, the virus attachment, penetration, and virucidal assays, and SARS-CoV-2 Mpro and PLpro activity assay were used to elucidate the mode of action. RESULTS: RDC exhibited dose-dependent inhibition of HCoV-OC43-induced cytopathic effects, reducing viral RNA copy numbers and viral protein levels. Time-of-addition assays indicated that RDC targets the early stages of the HCoV-OC43 life cycle, inhibiting virion attachment and penetration with virucidal activity. Notably, filixic acid ABA and dryocrassin ABBA, constituents of RDC, reduced HCoV-OC43 viral RNA loads. Furthermore, RDC effectively blocked viral entry in pseudotyped lentivirus assays, involving spike proteins of SARS-CoV-2 Delta plus and South Africa variants, as well as control lentiviral particles expressing vesicular stomatitis virus glycoprotein G. Additionally, RDC demonstrated inhibition of SARS-CoV-2 infection and its variants by targeting viral proteases, namely main protease (Mpro) and papain-like protease (PLpro). CONCLUSIONS: These findings underscore RDC's multistage approach to targeting viral infections by impeding virus entry and inhibiting viral protease activity. Therefore, RDC holds promise as a potent, broad-spectrum anticoronaviral therapeutic agent.

Anticoronaviral Activity of the Natural Phloroglucinols, Dryocrassin ABBA and Filixic Acid ABA from the Rhizome of Dryopteris crassirhizoma by Targeting the Main Protease of SARS-CoV-2.

The rhizome of Dryopteris crassirhizoma Nakai. (Dryopteridaceae) has been used in traditional medicine in East Asia and has recently been reported to have anticancer, anti-inflammation, and antibacterial activity as well as antiviral activity. Natural phloroglucinols from D. crassirhizoma, dryocrassin ABBA and filixic acid ABA were reported to inhibit influenza virus infection with an inhibitory activity on neuraminidase. In this study, we found that dryocrassin ABBA and filixic acid ABA have an inhibitory activity against the main protease of SARS-CoV-2. Therefore, dryocrassin ABBA and filixic acid ABA exhibited inhibitory activity against SARS-CoV-2 infection in Vero cells dose-dependently using the immunofluorescence-based antiviral assays. Moreover, these compounds inhibited SARS-CoV and MERS-CoV infection, suggesting their broad-spectrum anticoronaviral activity. In addition, a 5-day repeated-dose toxicity study of dryocrassin ABBA and filixic acid ABA suggested that an approximately lethal dose of these compounds in mice was >10 mg/kg. Pharmacokinetic studies of dryocrassin ABBA showed good microsomal stability, low hERG inhibition, and low CYP450 inhibition. In vivo pharmacokinetic properties of dryocrassin ABBA showed a long half-life (5.5-12.6 h) and high plasma exposure (AUC 19.3-65 µg·h/mL). Therefore, dryocrassin ABBA has therapeutic potential against emerging coronavirus infections, including COVID-19.

Dryocrassin ABBA ameliorates Streptococcus pneumoniae-induced infection in vitro through inhibiting Streptococcus pneumoniae growth and neutralizing pneumolysin activity.

To explore the role of dryocrassin ABBA (ABBA) in the prevention and treatment of Streptococcus pneumoniae (S. pneumoniae) infections in vitro, a minimal inhibitory concentration test, growth curve assay, hemolysis assay, BacLight LIVE/DEAD staining experiments, oligomerization inhibition assay, time-killing test, LDH release detection assay and cytotoxicity test were performed to evaluate the efficacy of ABBA against S. pneumoniae infections in vitro. The results indicated that ABBA treatment exists bactericidal effect on S. pneumoniae at a concentration of less than 8 µg/ml. Furthermore, ABBA was effective at inhibiting the oligomerization of pneumolysin (PLY) from reducing its hemolytic activity. Meanwhile, ABBA could ameliorate cell injury by neutralizing the biological activity of PLY without cytotoxicity. In summary, ABBA was a leading compound against S. pneumoniae infections through bactericidal effect and neutralizing PLY activity.

Total synthesis of dryocrassin ABBA and its analogues with potential inhibitory activity against drug-resistant neuraminidases.

The stems of Dryopteris crassirhizoma, one of the main components of Lianhua-Qingwen Formula (LQF) was traditionally used for heat-clearing and detoxifying. Dryocrassin ABBA is a key antiviral component in the herbal medicine while the compound is hard to get in large amounts with the features of homologous compounds, polyphenol groups, and low contents. Therefore, the present work aims to seek influenza H7N9 virus inhibitors from natural source by synthesis of dryocrassin ABBA and its analogues. As a result, total synthesis of the compound was achieved in nine steps with an over-all yield of 4.6%. Neuraminidases (NAs) inhibitory activities of the synthesized product and its analogues were evaluated afterward. Comparing with the positive control, OSV (9.6 µM), it was very exciting that dryocrassin ABBA and its analogues (b5 and e2) showed better NAs inhibitory activity against Anhui H7N9 with IC50 values of 3.6 µM, 2.5 µM and 1.6 µM. For the highly resistant Shanghai N9, these compounds can also show medium inhibitory activities. Docking results indicated the direct interaction of synthesized 3 hits with the key K294 by hydrogen bonds, but no direct interaction of OSV with the key K294 was observed in Shanghai N9. This study suggested that dryocrassin ABBA and its analogues especially AB, which consisted of polyphenol groups may have beneficial effects on treating avian influenza H7N9 virus.

An Inhibitory Effect of Dryocrassin ABBA on Staphylococcus aureus vWbp That Protects Mice From Pneumonia.

Von Willebrand factor-binding protein (vWbp), secreted by Staphylococcus aureus (S. aureus), can activate host prothrombin, convert fibrinogen to fibrin clots, induce blood clotting, and contribute to pathophysiology of S. aureus-related diseases, including infective endocarditis, staphylococcal sepsis and pneumonia. Therefore, vWbp is an promising drug target in the treatment of S. aureus-related infections. Here, we report that dryocrassin ABBA (ABBA), a natural compound derived from Dryopteris crassirhizoma, can significantly inhibit the coagulase activity of vWbp in vitro by directly interacting with vWbp without killing the bacteria or inhibiting the expression of the vWbp. Using molecular dynamics simulations, we demonstrate that ABBA binds to the "central cavity" in the elbow of vWbp by interacting with Arg-70, His-71, Ala-72, Gly-73, Tyr-74, Glu-75, Tyr-83, and Gln-87 in vWbp, thus interfering with the binding of vWbp to prothrombin. Furthermore, in vivo studies demonstrated that ABBA can attenuate injury and inflammation of mouse lung tissues caused by S. aureus and increase survival of mice. Together these findings indicate that ABBA is a promising lead drug for the treatment of S. aureus-related infections. This is the first report of potential inhibitor which inhibit the coagulase activity of vWbp by directly interacting with vWbp.

Anti-Influenza Virus (H5N1) Activity Screening on the Phloroglucinols from Rhizomes of Dryopteris crassirhizoma.

For screening the active phloroglucinols on influenza virus (H5N1) from Dryopteris crassirhizoma NaKai, a database was established including twenty-three phloroglucinols that had been isolated from Dryopteris crassirhizoma. Their inhibitory effect on the neuraminidase (NA) of influenza virus H5N1 was screened by molecular docking. As a result, three candidates were selected. The rhizomes of D. crassirhizoma were subjected to isolation and purification processes to obtain the inhibitor candidates. Thirteen phloroglucinols were obtained, including three selected candidates and two new phloroglucinols. The five phloroglucinols were investigated for their inhibitory activity on NA in vitro. The results showed that dryocrassin ABBA and filixic acid ABA exhibited inhibitory effects on NA with IC50 as 18.59 ± 4.53 and 29.57 ± 2.48 µM, respectively, and the other three phloroglucinols showed moderate inhibitory activity. Moreover, the anti-influenza virus (H5N1) activity and cytotoxicity of dryocrassin ABBA and filixic acid ABA were tested on Madin-Darby canine kidney (MDCK) cells with the cell counting kit-8 (CCK8) method. The results confirmed that dryocrassin ABBA exhibited an inhibitory activity with low cytotoxicity (TC50 > 400 µM) against influenza virus (H5N1) which will have to be investigated in further detail. In conclusion, phloroglucinols from D. crassirhizoma were shown to have anti-influenza virus activity, and especially dryocrassin ABBA, one of the phloroglucinols, may have the potential to control influenza virus (H5N1) infection.

Corrigendum: Dryocrassin ABBA, a novel active substance for use against amantadine-resistant H5N1 avian influenza virus.

[This corrects the article on p. 592 in vol. 6, PMID: 26136733.].

Dryocrassin ABBA, a novel active substance for use against amantadine-resistant H5N1 avian influenza virus.

The occurrence of multi-drug resistant highly pathogenic avian influenza virus (HPAIV) strains highlights the urgent need for strategies for the prevention and control of avian influenza virus. The aim of our current study is to evaluate the antiviral activity of dryocrassin ABBA isolated from Rhizoma Dryopteridis Crassirhizomatis (RDC) against an amantadine-resistant H5N1 (A/Chicken/Hebei/706/2005) strain in a mouse model. Post inoculation with HPAIV H5N1 virus in mice, the survival rate was 87, 80, and 60% respectively in the 33, 18, and 12.5 mg/kg dryocrassin ABBA-treated groups. On the other hand, the survival rate was 53 and 20%, respectively in the amantadine-treated group and untreated group. Mice administered with dryocrassin ABBA or amantadine showed a significant weight increase compared to the untreated group. Moreover, 33 and 18 mg/kg dryocrassin ABBA have decreased lung index (P >0.05) and virus loads (P <0.01) compared to the untreated group on day 7. Also, on day 7 bronchoalveolar lavage fluid pro-inflammatory cytokines (IL-6, TNF-α, and IFN-γ) decreased significantly (P <0.01) while anti-inflammatory cytokines (IL-10 and MCP-1) were increased significantly (P <0.01) in the 33 and 18 mg/kg dryocrassin ABBA-treated groups compared to the amantadine group and the untreated group. Moreover, the concentrations of IL-12 in drug-treated groups were significantly (P < 0.01) lowered compared with the untreated group. Based on the above we conclude that orally administered dryocrassin ABBA provided mice protection against avian influenza virus H5N1 by inhibiting inflammation and reducing virus loads. Dryocrassin ABBA is a potential novel lead compound which had antiviral effects on amantadine-resistant avian influenza virus H5N1 infection.

Application of a sensitive and accurate LC-MS/MS method for determination of dryocrassin ABBA in rat plasma for a bioavailability study.

A sensitive and accurate liquid chromatography-tandem mass spectrometry method was developed and validated for the determination of dryocrassin ABBA, a potential active component isolated from Dryopteris crassirhizoma, in rat plasma. Chromatographic separation was achieved on a Zorbax SB-C18 column (50 × 2.1 mm, 1.8 µm), with elution consisting of eluent (A) 10 mm ammonium acetate in methanol containing 0.1% formic acid and (B) 10 mm ammonium acetate in water containing 0.1% formic acid (A:B = 99:1, v/v) at a flow rate of 0.3 mL/min. Multiple reaction monitoring mode was used to monitor the precursor-product ion transitions of m/z 819.3 → 403.4 for dryocrassin ABBA and m/z 426.2 → 409.2 for internal standard. This assay exhibited a good linearity with a correlation coefficient >0.99 and showed no endogenous interference with the analyte and internal standard. The lower limit of quantification of dryocrassin ABBA was 4 ng/mL in 50 µL of rat plasma. The method was successfully applied in the pharmacokinetic study of dryocrassin ABBA in rats after intravenous (2.35 mg/kg) and oral (23.5 mg/kg) doses of dryocrassin ABBA. The oral bioavailability (F) of dryocrassin ABBA was estimated to be 50.1%. Our study is the first to clarify the pharmacokinetic behaviors of dryocrassin ABBA in animals.

Separation and purification of total poly phenol from Dryopteris crassirhizoma with anion exchange resin / 中草药

Objective: To optimize the separation and purification of the total poly phenol from Dryopteris crassirhizoma with anion- exchange resin. Methods: The rates of elution and absorption, contents of dryocrassin ABBA, and total ploy phenol were used as makers to optimize the purification conditions of total ploy phenol. Results: The extracting solution of D. crassirhizoma passed through 201×7 hydrogen-oxygen the anion-exchange resin column (column diameter-column height, 1:7) at the rate of 6 BV/h in reverse direction, then the resin column was flushed with water at the rate of 6BV/h to pH value 6-7 in forward direction. At last, the column was eluted at the rate of 6 BV/h to obtain ploy phenol with 9BV 3% salt water in 60% alcohol. The elution ratio of dryocrassin ABBA is 90.1%, and the total ploy phenol is 91.1%. The content of dryocrassin ABBA is 29.4% and the content of the total ploy phenol is 49.2%. Conclusion: The separation and purification of the total ploy phenol from D. crassirhizoma with 201 × 7 hydrogen-oxygen anion-exchange resin can achieve the satisfactory results which have wide application prospects.