Anti-inflammatory and antiviral activities of flavone C-glycosides of Lophatherum gracile for COVID-19

Graphical abstract

The emergence of SARS-CoV-2 variants has decreased the efficacy of vaccines (Callaway, 2022;Luo et al., 2022); however, the combined use of immunomodulators and antiviral agents is expected to have synergistic effects in the treatment of COVID-19 (Wang & Wang, 2022).Recent studies have demonstrated anti-inflammatory and antiviral effects in L. gracile.Ethanol extracts of L. gracile attenuate superoxide anion generation in stimulated neutrophils (Lai et al., 2021).Water extracts of L. gracile inhibit SARS-CoV-2 replication and decrease virusinduced cytotoxicity (Jan et al., 2021).However, the bioactive components in L. gracile have yet to be identified.Thus, this study investigated the potential effects of L. gracile in the treatment of COVID-19 as well as the bioactive constituents and underlying mechanisms of action.

Sample preparation and extraction
Dried L. gracile was purchased in January 2021 from Healing Herbar, a distributor of herbal remedies in Taipei, Taiwan.A voucher specimen of plant material (CGU-HTLTCM-40) was delivered to the herbarium of Chang Gung University (Taoyuan, Taiwan).L. gracile (2 g) was cut into 1 cm sections and placed in a Dionex ASE 350 auto-extraction device (Thermo Fisher Scientific, USA).The cells were filled with 60 ml of nhexane, ethyl acetate, methanol, or 50 % methanol.Extraction was performed twice at 50 • C for 15 min.Solvents were removed under vacuum to produce extracts of L. gracile, respectively referred to as LG-H, LG-EA, LG-M, or LG-50 M, in accordance with the extraction medium.

Fractionation of L. Gracile extracts using high-performance liquid chromatography (HPLC)
L. gracil methanol extract (LG-M) was dissolved in HPLC-grade methanol (10 mg/ml) and filtered through a 0.45 µm filter before being loaded for HPLC.Fractionation was conducted using a Shimadzu Nexera-I LC-2040C-3D HPLC system (Kyoto, Japan) with a Cosmosil 5C18-MS-II HPLC column (10 × 250 mm; Nacalai Tesque, Inc., Kyoto, Japan).The solvent system consisted of an aqueous solution and methanol with or without 0.1 % formic acid.Methanol gradient elution was performed under the following conditions: 50 % for 0-15 min and 50-100 % for 15-20 min.The flow rate was maintained at 5 ml/min, and the injection volume was 500 µl.Liquid chromatography was performed at 30 • C.

Analysis of L. Gracile extracts by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS)
Qualitative and quantitative analyses of L. gracile extracts were conducted using a Shimazu Nexera X2 UPLC system (Kyoto, Japan).
Liquid chromatography was performed using a YMC-Triart C18 (1.9 μm, 1.2 nm 50 mm × 2.1 mm) column (Kyoto, Japan).The mobile phase consisted of MeCN (A, containing 0.1 % formic acid) and water (W, containing 0.1 % formic acid).Gradient elution was performed using a flow rate of 0.5 ml/min and column temperature of 35 • C as follows: 8 % A for 0-12 min; 8-20 % A for 12-22 min; 20-100 % A for 22-28 min; 100 % A for 28-38 min.Samples were prepared by dissolving L. gracile extracts in methanol (2 mg/ml) and then filtered through a 0.45 μm filter.The sample volume was 1 μl per injection.Production scans (in positive and negative modes) and multiple reaction monitoring experiments (MRM) were conducted using a Shimazu LCMS-8045 mass spectrometry (Kyoto, Japan).The dwell time was 100 msec, and collision energy (CE) was optimized for individual compounds.All acquired MS data were processed using LCMS LabSolutions software (Version 5.93, Shimazu, Kyoto, Japan).
Non-targeted MS 1 and MS 2 data with m/z 100-2000 were collected (Zhang et al., 2021).MS data obtained via data-dependent acquisition (DDA) were analyzed using Waters MassFragment software (Mas-sLynx4.1,Waters, MA, USA).MS/MS molecular networking data were Y.-L.Chen et al. generated using a GNPS web-based platform (https://gnps.ucsd.edu).MS/MS spectra were filtered to identify the top 5 strongest ion peaks in the ± 50 Da window.A network was created based on linkages between molecular nodes filtered by cosine volume (0.7) and at least four matched peaks.The nodes that appeared in the network were annotated based on the experimental MS 2 fragmentation of isolates.The molecular network was visualized using Cytoscape 3.8.2(NRNB, CA, USA).

Measurement of 1 H nuclear magnetic resonance spectrum
Samples were dissolved in deuterium solvent at 5 mg/ml.Nuclear magnetic resonance (NMR) spectra were obtained using a Bruker AVANCE-400 MHz FT NMR spectrometer (MA, US).

Preparation of human neutrophils
Blood samples were drawn from healthy human donors (20-30 years old) under the approval and supervision of the Institutional Review Board of Chang Gung Memorial Hospital (202101115A3).Neutrophils were purified in accordance with a standard protocol involving dextran sedimentation, hypotonic lysis of erythrocytes, and Ficoll Hypaque gradient centrifugation, as described previously (Chiang, Cheng, et al., 2020).Isolated human neutrophils were evaluated by trypan blue assay, and > 98 % of living cells were preserved in HBSS (pH 7.4) under icecold conditions until use.

Assessment of superoxide anion generation
Superoxide anion generation was assessed using ferricytochrome c reduction.Briefly, neutrophils (6 × 10 5 cells/ml) were incubated with ferricytochrome c at 37 • C for 5 min.The cells were then incubated with the L. gracile extracts, pure compounds, or DMSO (0.1 %, as control) for 5 min.Neutrophils were treated using CB for 3 min followed by fMLF (0.1 μM) stimulation.Changes in absorbance with the reduction of ferricytochrome c were monitored continuously at 550 nm using a spectrophotometer (U-3010, Hitachi, Tokyo, Japan).

Assessment of lactate dehydrogenase (LDH) release
The cytotoxicity of neutrophils was evaluated in a cell-free medium in terms of the percentage of the total LDH release (Lai et al., 2021).Neutrophils (6 × 10 5 cells/ml) were equilibrated at 37 • C and then incubated for 5 min before treating samples for 15 min, after which the cells were lysed via Triton X-100 (0.1 %) for 30 min.The changes in absorbance at 490 nm were monitored continuously after adding LDH reagent.

Analysis of total ROS production
Total ROS production by neutrophils was measured using the luminol-amplified chemiluminescence method.Human neutrophils (2 × 10 6 cells/ml) were pre-mixed with 37.5 μM luminol and 6 U/ml horseradish peroxidase at 37 • C for 5 min.Cells were then incubated with DMSO or tested agents for 5 min, followed by stimulation with 0.1 μM fMLF.A 96-well chemiluminometer (Tecan Infinite F200 Pro; Männedorf, Switzerland) was used to detect changes in chemiluminescence.

Immunofluorescence staining of NETs
Human neutrophils were incubated on poly-L-lysine-coated glass coverslips at 37 • C for 30 min and then incubated with DMSO or tested agents for 10 min before stimulating the neutrophils through the addition of 10 nM PMA for 2 h.Cells were fixed with 4 % paraformaldehyde for 15 min and then treated with 5 % goat serum buffer for blocking.

Molecular docking
Ligand-protein interactions were investigated via in silico docking analysis using Discovery Studio 2019 (Biovia, Corp. CA, USA).The 3D structure of isoorientin was downloaded from PubChem (CID 114776).The crystal structure of SARS-CoV-2 spike protein was obtained from the RCSB Protein Data Bank (PDB ID: 6M0J).The initial spike and ligand structures were created using a CHARMm force field and minimized.A sphere radius covering the spike receptor-binding domain was set.Docking poses were generated in accordance with the CDOCKER protocol using Discovery Studio software.

Statistics
All experiment results were expressed as mean ± S.E.M., and the Student's t-test was used for statistical analysis via GraphPad Prism software (GraphPad Software, San Diego, CA, USA).Differences with p values < 0.05 were considered statistically significant.

L. Gracile methanol extract (LG-M) attenuates superoxide anion generation in fMLF-activated human neutrophils
Neutrophilic inflammation is triggered by pathogen-associated molecular patterns, such as fMLF.Excessive superoxide anions produced by activated neutrophils cause tissue damage during inflammation.L. gracile is an edible plant used for centuries as a functional food and herbal medicine.Previous studies have shown that L. Gracile ethanol extracts attenuate superoxide anion generation and neutrophil activity (Lai et al., 2021).To identify the active constituents, we first sought to Y.-L.Chen et al. clarify the general polar properties of active components.L. gracile was extracted using n-hexane, ethyl acetate, methanol, or 50 % methanol to create extracts with diverse polar properties.Only the L. gracile methanol extract (LG-M) inhibited superoxide anion generation in fMLFactivated human neutrophils (Fig. S1A).Note that LG-M did not affect the viability of neutrophils in an LDH assay, indicating that the observed reduction in superoxide anion production was not due to cytotoxic effects (Fig. S1B).

Flavone C-glycosides of L. Gracile restrict respiratory bursts in stimulated neutrophils
Flavone C-glycosides are a major group of chemical components in L. gracile.Isoorientin is generally used in the quality control of L. gracile products (Fan, Lee, & Lin, 2015;Wang et al., 2012); however, the roles of flavone C-glycosides in neutrophilic inflammation have seldom been addressed.To elucidate the anti-inflammatory effects of flavone C-glycosides of L. gracile, a flavonoid-enriched extract from LG-M (FlavoLG) was produced using a simple process based on liquid chromatography (Fig. 1A).The structural features of FlavoLG were elucidated via NMR and MS 2 -based molecular networking experiments.The 1 H NMR spectrum presented proton signals characteristic of C-glycosides and substituted aromatic rings (Hao et al., 2016) (Fig. 1B).MS 2 data revealed that flavone C-glycosides made up the largest molecular cluster.The high yellow-to-green area ratio in the clustering nodes indicates a high concentration of flavone C-glycosides in FlavoLG (Fig. 1C).Isoorientin was then quantified using optimized MRM experiments and used as makers representing flavone C-glycosides in FlavoLG (Fig. S2).The amount of isoorientin in FlavoLG was 5.98 %.These qualitative and quantitative results demonstrated the efficacy of methanolic chromatography in the creation of extracts rich in flavone-C-glycosides from L. gracile.Bioassay results revealed that FlavoLG and flavone-C-glycosides (isoorientin and orientin) reduced superoxide anion generation in neutrophils at the concentration without cytotoxicity, as indicated by IC 50 values of 7.02 ± 0.56 µg/ml, 6.56 ± 0.17 µM, and 9.57 ± 0.41 µM (Fig. 2 and Fig. S3).Furthermore, FlavoLG, isoorientin, and orientin significantly reduced the production of ROS, as indicated by IC 50 values of 0.17 ± 0.01 µg/ml, 0.16 ± 0.00 µM, and 0.15 ± 0.01 µM (Fig. 3).Superoxide anion can be converted to ROS.Our data suggest that Fla-voLG and two major flavone C-glycosides, orientin and isoorientin, are more sensitive to inhibit ROS generation (Fig. 3) than superoxide production (Fig. 2), suggesting that FlavoLG, orientin, and isoorientin decrease neutrophil ROS formation via direct scavenging and indirect regulation of cell responses.Indeed, the potent ROS scavenging activity is attributed by the high electron resonance property of phenoxyl substructure in flavonoids (Zhang, et al., 2015).These results indicate that flavone-C-glycosides of L. gracile reduce respiratory bursts in activated neutrophils.

Flavone C-glycosides of L. Gracile inhibit the formation of neutrophil extracellular traps (NETs) induced by phorbol 12-myristate 13-acetate (PMA)
NETs are web-like structures consisting mainly of nuclear chromatin coated with granular proteins of neutrophils, such as neutrophil elastase (McKenna et al., 2022).NETs play an important role in inflammatory and autoimmune disorders, such as rheumatoid arthritis, atherosclerosis, and diabetes (Huang et al., 2022;Mutua & Gershwin, 2021).Excessive NET levels can cause tissue damage and enhance recruitment of immune cells into inflammatory sites (Wang, Du, Hawez, Mörgelin, & Thorlacius, 2019).Inhibiting unregulated NET formation could have beneficial effects in dealing with neutrophil-associated diseases.In this study, FlavoLG and flavone C-glycosides were found to decrease PMAinduced NET levels (Fig. 4A and 4B) in immunofluorescence staining (Fig. 4C).Superoxide anions and ROS associated with respiratory bursts were shown to play crucial roles in NOX-dependent NETs (Azzouz et al., 2021;Feitz et al., 2021).Our results revealed that the reduction in NET levels by flavone C-glycosides can be attributed to restrictions on respiratory bursts of activated neutrophils.

Flavone C-glycosides of L. gracile decrease SARS-CoV-2 infection by interfering with the binding of SARS-CoV-2 spike and angiotensinconverting enzyme 2 (ACE2)
Flavonoids are considered as safe and promising agents against COVID-19 for their antioxidant, antiviral, and anti-inflammatory activities (Alzaabi et al., 2022).Several flavones and flavone O-linked glycosides have been demonstrated to inhibit SARS-CoV-2 via targeting Mpro, 3CLpro, or RdRp in in silico and in vitro studies (Alzaabi et al., 2022;Jantan et al., 2022).Discovering SARS-CoV-2 spike blockers in natural products is a solid strategy for the prevention and/or treatment of SARS-CoV-2 infection (Tomas et al., 2022).Several dietary polyphenols have been shown to affect the interactions between the SARS-CoV-2 spike and ACE2 (Schmidt, Hakeem Said, Ohl, Sharifii, Cotrell, & Kuhnert, 2022).However, the role of flavone C-glycosides in spike/ ACE2 interaction is rarely discussed.L. gracile has been shown to inhibit SARS-CoV-2 replication and decrease cytotoxicity (Jan et al., 2021); however, the active constituents have yet to be identified.In the current study, FlavoLG and isoorientin inhibited infection of SARS-CoV-2 pseudovirus variants possessing D614G or Omicron spikes into hACE-2-overexpressed HEK293T cells (Fig. 5A) but did not induce cytotoxicity in hACE-2-overexpressed HEK293T cells (Fig. S4).The SARS-CoV-2 spike receptor binding domain (RBD) binds to the ACE2 N-terminal peptidase domain through interactions with key amino acids.Residues Y449 and K417 of spike RBD facilitate ACE2 recognition by forming hydrogen bonds and salt bridges (Lan et al., 2020).Molecular docking analysis was performed to determine whether flavone C-glycosides inhibit viral infection by affecting spike/ACE2 binding.Docking result revealed that isoorientin interacts with Y449, K417, and other amino acids of spike-RBD (CDOCKER energy − 27.19 kcal/mol) (Fig. 6).The blockage between SARS-CoV-2 spike and ACE2 was further evaluated using FRET assays.FlavoLG and isoorientin were shown to reduce spike/ ACE2 binding in a dose-dependent manner (Fig. 5B).Our data indicated that flavone C-glycosides of L. gracile are the antiviral components inhibiting SARS-CoV-2 infection.Note that isoorientin and other flavone C-glycosides of L. gracile also inhibit the respiratory syncytial virus (Chen et al., 2019;Wang et al., 2012).The broad antiviral spectrum of L. gracile extracts could greatly expand the applicability of this compound in the development of antiviral products.Most flavonoids have low oral bioavailability; therefore the in vivo anti-inflammatory effect and anti-SARS-CoV-2 infection activity require further study before clinical application.

Conclusions
This study demonstrated that flavone C-glycosides of L. gracile are active components against neutrophilic inflammation and SARS-CoV-2 infection.This study also developed a chromatography-based method for the extraction of flavone C-glycosides.These results provide scientific evidence indicating the efficacy of L. gracile as a potential supplement for the prevention of COVID-19 and neutrophil-associated disorders.
The authors confirm that there are no conflicts of interest associated with this study.Y.-L.Chen et al.

Fig. 1 .
Fig. 1.Structural characteristics of L. gracile flavonoid-enriched extract (FlavoLG).(A) Indicated fraction of LG-M was collected in the form of FlavoLG using a liquid chromatography-based method involving methanolic gradient elution without the addition of formic acid.Colored chromatography results revealed UV absorption patterns typical of coumaroylquinic acids (CoQAs) at 310 nm (red) and flavonoids at 350 nm (blue); (B) Blue arrows indicate proton signals indicative of flavone C-glycosides in 1 H NMR spectra, involving protons on C-glycosides and substituted aromatic rings; (C) Combined molecular networks of FlavoLG and LG-M.According to MS 2 fragmentation analysis, flavone C-glycosides made up the largest cluster.The colored area of each node indicates the relative quantities of given phytochemicals in FlavoLG (yellow) and LG-M (green).Flavone C-glycoside levels were higher in FlavoLG than in LG-M; (D) Structural illustration showing selected flavone C-glycosides in FlavoLG.

Fig. 5 .
Fig. 5. Flavone C-glycosides of L. gracile inhibit SARS-CoV-2 pseudovirus infection by interfering with spike/ACE2 binding.(A) SARS-CoV-2 pseudovirus was pretreated with DMSO (0.1 %) or samples and then infected hACE-2-overexpressed HEK293T for 1 h.The infected cells were washed and incubated with fresh medium until luciferase activity was measured using a Luciferase Assay kit; (B) Tag1-SARS-CoV-2 spike protein and Tag2-ACE2 protein were pretreated with FlavoLG or isoorientin to which anti-tags were added.Spike/ACE2 binding was evaluated by measuring the triggered FRET at 665 nm.The data are presented as mean ± S.E.M. (n = 3).** p < 0.01 and *** p < 0.001, compared with the DMSO group.