Lonicerae Japonicae Flos Attenuates Neutrophilic Inflammation by Inhibiting Oxidative Stress

Lonicerae japonicae flos (LJ) is an Asian traditional herb that is used as a dietary supplement, tea, and beverage to clear heat and quench thirst. However, no studies investigated its effect on activated human neutrophils, which played a crucial role in the bad prognosis of coronavirus disease of 2019 (COVID-19) patients by aggravating lung inflammation and respiratory failure. Herein, we evaluated the anti-inflammatory effect of LJ ethanol extract (LJEE) on human neutrophils activated by N-formyl-methionyl-leucyl-phenylalanine (fMLF). Our experimental results indicated that LJEE suppressed fMLF-activated superoxide anion (O2•−) generation, the expression of CD11b, and cell adhesion and migration, as well as the formation of neutrophil extracellular traps in human neutrophils. Further in-depth mechanical investigation revealed that pretreatment with LJEE accelerated the Ca2+ clearance, but did not affect the phosphorylation of mitogen-activated protein kinases (MAPKs) and protein kinase B (Akt) in activated human neutrophils. In addition, LJEE displayed a dose-dependent reactive oxygen species (ROS) scavenger activity, which assisted its anti-inflammatory activity. From the bioassay-coupled chromatographic profile, chlorogenic acids were found to dominate the anti-inflammatory effects of LJEE. Moreover, LJ water extract (LJWE) demonstrated an interrupting effect on the severe acute respiratory syndrome coronavirus-2 spike protein (SARS-CoV-2-Spike)/angiotensin-converting enzyme 2 (ACE2) binding. In conclusion, the obtained results not only supported the traditional use of LJ for heat-clearance, but also suggested its potential application in daily health care during the COVID-19 pandemic.

ysis. To identify the main active constituents, we developed a bioassay-coupled highperformance liquid chromatography (HPLC) approach to construct the LJEE biochemical profile. The anti-inflammatory constituents of LJEE were identified and were further quantified using a multiple reaction monitoring tandem mass spectrometric approach (MRM-MS/MS). Calcium mobilization modulated the anti-inflammatory activity of LJEE. This study illustrated the biological activity of LJEE in targeting neutrophil-dominant inflammatory diseases. We also investigated the effects of LJ water extract (LJWE) on the homogeneous time-resolved fluorescence (HTRF) SARS-CoV-2 Spike/angiotensin-converting enzyme 2 (ACE2) binding assay to validate the suggested activity against COVID-19.

Preparation of Human Neutrophils
Human donors (20-35 years old) provided blood by venipuncture. The protocol was approved and supervised by the Institutional Review Board (IRB) at Chang Gung Memorial Hospital. The purification of neutrophils was achieved according to a previously reported method [29]. The protocol involved dextran sedimentation, hypotonic lysis, and Ficoll Hypaque gradient of erythrocytes. After the isolation of human neutrophils, they were suspended in a 50 mL centrifuge tube in a calcium (Ca 2+ )-free and magnesium (Mg 2+ )-free HBSS buffer (KH 2 PO 4 : 60 mg/L; KCl: 400 mg/L; NaCl: 8000 mg/L; NaHCO 3 : 350 mg/L; dextrose: 1000 mg/L). The pH was adjusted to 7.4, and the trypan blue exclusion method was used to examine the neutrophils (>98% viable cells). The assessment of the neutrophils was achieved in HBSS (1 mM CaCl 2 contained) at 37 • C.

Fraction Preparation for Bioassay-Coupled High-Performance Liquid Chromatography (HPLC) Profile
A Shimazu preparative HPLC system (Shimazu, Kyoto, Japan) was used for profiling the LJEE extract. A COSMOSIL 5C18-MS-II Waters (20 × 250 mm, C18) column (Nacalai Tesque, Kyoto, Japan) was used for the liquid chromatography experiments. Methanol (MeOH, A) and water (W, containing 0.1% formic acid) were mixed and were used as the mobile phase as follows: 0-30 min, 10-60% A; 30-60 min, 60-80% A; 60-70 min, 80-100% A; 70-85 min, 100% A. The temperature of the column was maintained at 35 • C, the mobile phase flow rate was fixed at 10 mL/min, and compounds were detected at wavelengths from 190 to 500 nm. The fractionation sample was prepared using a 1 mg drug sample dissolved in 10 µL of methanol that was filtered using a 0.45 µm membrane filter, and then was loaded into the column. The sample injection was applied manually (100 µL volume per injection). Fractions were collected every 5 min of the retention time window. The collected fractions were subjected to a bioassay.

Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry (UPLC-MS/MS) Condition
A qualitative and quantitative analysis of LJEE extract was performed using a Shimazu NexeraX2 UPLC (Shimazu, Kyoto, Japan). A Thermo Hypersil GOLD C18 (1.9 µm, 2.1 mm × 100 mm) column (Waltham, MA, USA) was used for the liquid chromatography experiments. MeOH (A) and water (W, containing 0.1% formic acid) were mixed and were used as follows: 0-5 min, 10% A; 5-20 min, 10-40% A; 20-30 min, 10% A. The temperature of the column was maintained at 35 • C, and the flow rate was fixed at 0.5 mL/min. For the sample preparation, 1 mg LJEE extract was dissolved in methanol (1 mL) and was filtered using a 0.45 µm filtering membrane, and then loaded into the column. Then, the sample injection (1 µL volume per) was applied automatically.
The mass spectrometer Shimazu LCMS-8045 was used for product ion scanning (positive or negative mode) and multiple reaction monitoring (MRM). At 100 msec, the dwell time was set, and then the collision energy was automatically optimized for each compound individually. LCMS LabSolutions software (Version 5.93, Shimazu, Kyoto, Japan) was used to process the obtained MS.

Determination of Lactate Dehydrogenase (LDH) Release
A cell-free medium was used to assess the cytotoxicity against neutrophils as the ratio of the LDH released in total [28]. At 37 • C, the neutrophils (6 × 10 5 cells/mL) were equilibrated and were incubated for 5 min before treatment with the LJEE extract (1, 3, 10, and 30 µg/mL). The treatment with the extract lasted 15 min. The cells were then Antioxidants 2022, 11, 1781 5 of 17 lysed using Triton X-100 (0.1%) at 25 • C for 30 min. The LDH reagent was added, and any changes in the absorbance at 492 nm (in 4.5 mL cuvette) were continuously monitored.

Determination of Superoxide Anion (O 2 •− ) Scavenging Activity
A cell-free system was used to assess the O 2 •− scavenging ability of LJEE based on examining the WST-1 reduction [28] in xanthine/xanthine oxidase. To the assay buffer [50 mM Tris (pH 7.4)], 0.02 U/mL xanthine oxidase and 0.3 mM WST-1 were added, followed by the addition of xanthine (0.1 mM) at 30 • C for 10 min. LJEE extract (1, 3, 10, and 30 µg/mL) or SOD (20 U/mL) was added to react with xanthine oxidase for 3 min. A spectrophotometer (U-3010, Hitachi, Tokyo, Japan) was used to measure the correlative absorbance of the O 2 •− -induced WST-1 reduction. The measurement was performed at 450 nm (in 4.5 mL cuvette).

Determination of Neutrophils Adhesion
Hoechst 33342 (1 ng/mL) was used to label neutrophils (5 × 10 6 cells/mL), and the temperature was maintained at 37 • C. The LJEE extract (30 µg/mL) was added to the labeled neutrophils for another 5 min [28]. At 37 • C, fMLF (0.1 µM)/CB (1 µg/mL) was used to activate the neutrophils. They were then co-cultured with bEnd.3 endothelial cells (ECs) on a 12-well plate for 30 min. To fix the cells, 4% paraformaldehyde was added. The counts of the neutrophils adhering to bEnd.3 cells were counted using a fluorescent microscope (Olympus Corporation, Center Valley, PA, USA).

Neutrophils Chemotactic Migration Assay
At 37 • C, the 5 × 10 6 cells/mL of incubated neutrophils were treated with LJEE extract (30 µg/mL) or DMSO for 5 min [28]. Then, the suspension of neutrophils was added to a Millicell Culture Plate Insert (pore size 3 µm) (Millipore Darmstadt, Germany). Then, the inserts were put into the dishes (which contained a chemokine solution). Ethylenediaminetetraacetic acid (EDTA) was added after 90 min. The counts of the migrated neutrophils were counted using a fluorescent microscope (Olympus Corporation, Center Valley, PA, USA).

Measurement of [Ca 2+ ] i
At 37 • C, the neutrophils (6 × 10 6 cells/mL) and fluo-3/AM (2 µM) were incubated for 30 min [28]. The cells were centrifuged (200 g) for 8 min at 4 • C. The pellets were acquired to resuspend in HBSS. At 37 • C, the solution of HBSS suspension was then incubated (for 3 min), followed by treatment with LJEE extract (10 and 30 µg/mL). The fMLF (0.1 µM) was added for neutrophil activation. Triton X-100 and ethylene glycol-bis(2-aminoethylether)-N,N,N ,N -tetraacetic acid (EGTA) were added in order to reach the maximum (F max ) and minimum (F min ) fluorescence values, respectively. A spectrofluorometer was used to detect the fluorescence intensity variations at 488 and 520 nm (in 4.5 mL cuvette).

Western Blotting
At 37 • C, the human neutrophils were pretreated with LJEE for 5 min. The cells were activated by fMLF (0.1 µM)/CB (1 µg/mL) [28]. At 100 • C, a sample buffer (62.5 mM pH 6.8 Tris-HCl, 4% sodium dodecyl sulfate, 5% β-mercaptoethanol, 0.0125% bromophenol blue, 8.75% glycerol, 1% protease inhibitor cocktail, and 1% phosphatase inhibitor cocktail) was used to quench the reaction for 15 min. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to separate the cell lysates. Western blotting was used with the relevant primary antibodies (MAPKs and Akt) and with horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h. The protein levels were then detected by an enhanced chemiluminescence system and a densitometer (UVP, Upland, CA, USA).

Statistics
For Figures 1, 2B, 3B and 6, one-way ANOVA and Dunnett's multiple comparison tests were employed. For Figure 2D-E, Figures 4 and 5, the Tukey test was used. GraphPad Prism software (GraphPad Software version 9, San Diego, CA, USA) was used for all statistical calculations. p values < 0.05 were considered to show statistically significant effects.

Lonicerae Japonicae Flos Ehanol Extract (LJEE) Inhibited the Superoxide Anion (O 2 •− ) Generation but Not Elastase Release in N-Formyl-Methionyl-Leucyl-Phenylalanine (fMLF)-Activated Human Neutrophils
Pathogen-associated molecular patterns (PAMPs) such as fMLF can activate neutrophils, resulting in a series of inflammatory responses, including degranulation (elastase release), respiratory burst (O 2 •− generation), chemotactic adhesion, and migration [32]. To evaluate the anti-inflammatory activity of LJEE extract, we first assessed its effect on respiratory burst and degranulation. Only O 2 •− generation, but not elastase release, was suppressed by the treatments with LJEE (3, 10, and 30 µg/mL) in fMLF-induced human neutrophils (IC 50 value of 10.16 ± 1.27 µg/mL) ( Figure 1A,B). LJEE extract did not show any cytotoxicity on human neutrophils, as demonstrated by the LDH release assay (data not shown). These findings suggested that the therapeutic potential of LJEE extract on neutrophilic inflammation is not due to the cytotoxic effect.
Respiratory burst plays a predominant role in defending foreign pathogens in activated neutrophils. The attenuation of human neutrophils oxidative stress-dependent inflammatory responses is considered a sign of the suppression of respiratory burst [33]. We used the cell-free xanthine/xanthine oxidase system and DPPH assay to evaluate the scavenging effect of LJEE extract against O 2 •− (a reactive oxygen species) or other free radicals of reactive nitrogen species. The treatment with LJEE did not scavenge O 2 •− , but it captured reactive nitrogen species ( Figure 1C,D). The LJEE extract dose-dependently scavenged free radicals in the DPPH assay. We found that the LJEE-scavenging property in activated human neutrophils was mediated through intracellular signaling modulation. The antioxidant property of LJEE on reactive nitrogen species may assist its anti-inflammatory activity. trophils (IC50 value of 10.16 ± 1.27 μg/mL) ( Figure 1A,B). LJEE extract did not show any cytotoxicity on human neutrophils, as demonstrated by the LDH release assay (data not shown). These findings suggested that the therapeutic potential of LJEE extract on neutrophilic inflammation is not due to the cytotoxic effect.
Respiratory burst plays a predominant role in defending foreign pathogens in activated neutrophils. The attenuation of human neutrophils oxidative stress-dependent inflammatory responses is considered a sign of the suppression of respiratory burst [33]. We used the cell-free xanthine/xanthine oxidase system and DPPH assay to evaluate the scavenging effect of LJEE extract against O2 •− (a reactive oxygen species) or other free radicals of reactive nitrogen species. The treatment with LJEE did not scavenge O2 •− , but it captured reactive nitrogen species ( Figure 1C,D). The LJEE extract dose-dependently scavenged free radicals in the DPPH assay. We found that the LJEE-scavenging property in activated human neutrophils was mediated through intracellular signaling modulation. The antioxidant property of LJEE on reactive nitrogen species may assist its anti-inflammatory activity.  In previous reports, the antioxidant activity of LJ was documented [34], and its free radical-scavenging activity was attributed to chlorogenic acid, a major constituent of LJEE [35]. The potent antioxidant effect suggested the potential therapeutic application of LJEE against oxidative stress-dependent inflammatory diseases. We found that LJEE's biological effect supports its use as an anti-inflammatory remedy and daily health supplement for well-being.

LJEE Suppressed NET Formation
NET formation participates in neutrophilic inflammatory disorders [29]. In our current study, NETs were released after stimulation with PMA (10 nM) for 3 h. In Figure 3, LJEE treatment reduced extracellular DNA structure formation, suggesting that the neutrophilic inhibitory effect of LJEE was achieved through the inhibition of NET formation.

The Phosphorylation of Mitogen-Activated Protein Kinases (MAPKs) and Protein Kinase B (Akt) Signaling in Activated Human Neutrophils Were Not Affected by LJEE Treatment
The intracellular signal pathways, such as MAPKs and Akt, were found to play critical roles during neutrophilic inflammation, involved in degranulation, oxidative burst, chemotaxis, and NET formation [37,38]. In order to evaluate the pharmacological effects of LJEE, the activation of Akt and MAPKs were assessed using the Western blotting approach. The pretreatment with LJEE (30 μg/mL) did not affect the fMLF-induced MAPKs and Akt phosphorylations in human neutrophils (Figure 4). Based on these findings, we suggested that the anti-inflammatory effects of LJEE might proceed through mechanisms other than MAPK and Akt signaling pathways.

The Phosphorylation of Mitogen-Activated Protein Kinases (MAPKs) and Protein Kinase B (Akt) Signaling in Activated Human Neutrophils Were Not Affected by LJEE Treatment
The intracellular signal pathways, such as MAPKs and Akt, were found to play critical roles during neutrophilic inflammation, involved in degranulation, oxidative burst, chemotaxis, and NET formation [37,38]. In order to evaluate the pharmacological effects of LJEE, the activation of Akt and MAPKs were assessed using the Western blotting approach. The pretreatment with LJEE (30 µg/mL) did not affect the fMLF-induced MAPKs and Akt phosphorylations in human neutrophils (Figure 4). Based on these findings, we suggested that the anti-inflammatory effects of LJEE might proceed through mechanisms other than MAPK and Akt signaling pathways.

LJEE Decreased the fMLF-Induced Neutrophilic Intracellular Ca 2+ Mobilization
In neutrophils, Ca 2+ is a major secondary messenger that participates in respiratory bursts, degranulation, and cytoskeleton rearrangements. The ligation of fMLF to formyl peptide receptor 1 (FPR1) induces PLC-associated phosphatidylinositol 4,5-bisphosphate (PIP 2 )-inositol triphosphate (IP 3 ) hydrolysis. After hydrolysis, it attaches to the IP 3 receptors on the endoplasmic reticulum (ER), where this binding initiates calcium mobilization from the ER to the cytoplasm [39]. Under physiological conditions, the activation of fMLF leads to a transient elevation in the intracellular calcium levels [Ca 2+ ]i up to a maximum level, which withdraws [Ca 2+ ]i to equilibrium in the aftermath within a short time (one minute). Our current results revealed that the pretreatment with LJEE extract (3 and 30 µg/mL) did not affect the level of the [Ca 2+ ] i peak ( Figure 5A,B), but reduced the time demanded for [Ca 2+ ] i to backtrack to half of its peak height (t 1/2 ; Figure 5B). It has been documented that the pharmacological inhibition of t 1/2 of calcium mobilization, but not the [Ca 2+ ] peak, still successfully alleviates neutrophil activation, including O 2 •− and ROS generation from respiratory burst [40][41][42]. Moreover, the restoration of Ca 2+ homeostasis was proved to be essential for the prevention of Ca 2+ overload and cell hyperactivity following the activation of neutrophils. This could be achieved through the rapid clearance of Ca 2+ by the unified operation of the plasma membrane Ca 2+ -ATPase and endo-membrane Ca 2+ -ATPase [43]. Thus, we suggested that LJEE extract exhibited neutrophilic inhibitory properties by accelerating the Ca 2+ clearance, and thus affects O 2 •− generation in a respiratory burst.

LJEE Decreased the fMLF-Induced Neutrophilic Intracellular Ca 2+ Mobilization
In neutrophils, Ca 2+ is a major secondary messenger that participates in respiratory bursts, degranulation, and cytoskeleton rearrangements. The ligation of fMLF to formyl peptide receptor 1 (FPR1) induces PLC-associated phosphatidylinositol 4,5-bisphosphate (PIP2)-inositol triphosphate (IP3) hydrolysis. After hydrolysis, it attaches to the IP3 receptors on the endoplasmic reticulum (ER), where this binding initiates calcium mobilization from the ER to the cytoplasm [39]. Under physiological conditions, the activation of fMLF leads to a transient elevation in the intracellular calcium levels [Ca 2+ ]i up to a maximum level, which withdraws [Ca 2+ ]i to equilibrium in the aftermath within a short time (one minute). Our current results revealed that the pretreatment with LJEE extract (3 and 30 μg/mL) did not affect the level of the [Ca 2+ ]i peak ( Figure 5A,B), but reduced the time demanded for [Ca 2+ ]i to backtrack to half of its peak height (t1/2; Figure 5B). It has been documented that the pharmacological inhibition of t1/2 of calcium mobilization, but not the [Ca 2+ ] peak, still successfully alleviates neutrophil activation, including O2 •− and ROS generation from respiratory burst [40][41][42]. Moreover, the restoration of Ca 2+ homeostasis was proved to be essential for the prevention of Ca 2+ overload and cell hyperactivity following the activation of neutrophils. This could be achieved through the rapid clearance of Ca 2+ Store-operated calcium entry (SOCE) was shown to play a critical role in NADPH oxidase regulation, which might produce a burst of O 2 •− , resulting in oxidative stress and the further development of inflammation [44]. α-PKC (α-protein kinase C), an element mediated by SOCE, was found to be a selective element in the positive signaling of fMLFinduced superoxide anion generation without affecting elastase release [45]. Therefore, we suggested that the LJEE-mediated intracellular calcium inhibition might selectively attenuate the generation of superoxide anion, but not the concurrent release of elastase, in human neutrophils ( Figures 1A,B and 5).
Antioxidants 2022, 11, x FOR PEER REVIEW 12 of 18 by the unified operation of the plasma membrane Ca 2+ -ATPase and endo-membrane Ca 2+ -ATPase [43]. Thus, we suggested that LJEE extract exhibited neutrophilic inhibitory properties by accelerating the Ca 2+ clearance, and thus affects O2 •− generation in a respiratory burst. Store-operated calcium entry (SOCE) was shown to play a critical role in NADPH oxidase regulation, which might produce a burst of O2 •− , resulting in oxidative stress and the further development of inflammation [44]. α-PKC (α-protein kinase C), an element mediated by SOCE, was found to be a selective element in the positive signaling of fMLFinduced superoxide anion generation without affecting elastase release [45]. Therefore, we suggested that the LJEE-mediated intracellular calcium inhibition might selectively attenuate the generation of superoxide anion, but not the concurrent release of elastase, in human neutrophils ( Figures 1A,B and 5).

Chlorogenic Acids Derivatives Dominated the Neutrophilic Inhibition of LJEE in fMLF-Induced Human Neutrophils
We further investigated the activity of phytoconstituents as an important requirement for the registration of functional food and herbal medications by regulatory authorities. We first constructed an anti-inflammatory assay-based high-performance liquid chromatography (HPLC) profile to interpret the relationships between the retention time (tR)-dependent fractions of LJEE and their activity on superoxide anion generation. As shown in the profile ( Figure 6A), the most potent anti-inflammatory fraction (tR: 34.3-39.5 min) exhibited 86.44% inhibition of O2 •− generation at 10 μg/mL. The isolation, purification, and structure elucidation led to the identification of three major chlorogenic acid derivatives from LJEE, chlorogenic acid (1), neochlorogenic acid (2), and cryptochlorogenic acid (3) ( Figure 6C). Among all the isolates, chlorogenic acid (1) is considered the major bioactive constituent, as reported in the "Pharmacopoeia of the People's Republic of China (Pharmacopoeia Commission of the Ministry of Public Health, People's Republic

Chlorogenic Acids Derivatives Dominated the Neutrophilic Inhibition of LJEE in fMLF-Induced Human Neutrophils
We further investigated the activity of phytoconstituents as an important requirement for the registration of functional food and herbal medications by regulatory authorities. We first constructed an anti-inflammatory assay-based high-performance liquid chromatography (HPLC) profile to interpret the relationships between the retention time (t R )-dependent fractions of LJEE and their activity on superoxide anion generation. As shown in the profile ( Figure 6A), the most potent anti-inflammatory fraction (t R : 34.3-39.5 min) exhibited 86.44% inhibition of O 2 •− generation at 10 µg/mL. The isolation, purification, and structure elucidation led to the identification of three major chlorogenic acid derivatives from LJEE, chlorogenic acid (1), neochlorogenic acid (2), and cryptochlorogenic acid (3) ( Figure 6C). Among all the isolates, chlorogenic acid (1) is considered the major bioactive constituent, as reported in the "Pharmacopoeia of the People's Republic of China (Pharmacopoeia Commission of the Ministry of Public Health, People's Republic of China, 2020)" and "Taiwan Herbal Pharmacopeia (Ministry of Health and Welfare, Taiwan, 2019)". Since the commercially available cynaroside (4) was also reported as a component of Lonicerae japonicae flos according to the Pharmacopoeia of the People's Republic of China, compounds 1-4 were assayed.
The obtained major compounds (1-4) from the active fraction of LJEE were evaluated for their effects on respiratory burst and degranulation. The chlorogenic derivatives (1-3) showed identical activities on superoxide anion, with IC 50 values ranging from 3.76 to 3.97 µM, but neochlorogenic acid (3) did not affect the elastase release at 20 µM (Table 1). However, the effect of cynaroside (4) on activated neutrophils seemed to be negligible, with 52.63% inhibition at 20 µM. The results of the chlorogenic derivatives (1-3) showed a similar bioassay profile to LJEE, with the significant suppression of superoxide anion scavenging, but not of elastase release. of China, 2020)" and "Taiwan Herbal Pharmacopeia (Ministry of Health and Welfare, Taiwan, 2019)". Since the commercially available cynaroside (4) was also reported as a component of Lonicerae japonicae flos according to the Pharmacopoeia of the People's Republic of China, compounds 1-4 were assayed. The obtained major compounds (1-4) from the active fraction of LJEE were evaluated for their effects on respiratory burst and degranulation. The chlorogenic derivatives (1-3) showed identical activities on superoxide anion, with IC50 values ranging from 3.76 to 3.97 μM, but neochlorogenic acid (3) did not affect the elastase release at 20 μM (Table 1). However, the effect of cynaroside (4) on activated neutrophils seemed to be negligible,  The qualitative and quantitative analyzing protocols were established to detect the exact amount of those compounds (1)(2)(3)(4) in LJEE extract. Tandem MS shows the high efficiency of compound characterization and quantitative analysis, in particular, the multiple reaction monitoring (MRM) experiment that is designed to detect the specific MS/MS fragmentations from the precursor compound ions based on a multi-quadrupole MS spectroscopy [46]. We hereby performed an MRM experiment on a triple quadrupole MS spectroscopy in order to come up with a rapid and sensitive method for the qualitative and quantitative quantification of the phytoconstituents of LJEE (Table 2, Figure 6B). The optimal detection of these compounds was selected to be the negative (−) mode, and the individual collision energies were optimized to give rise to the maximum response of their daughter ions. The characteristic product ions of each compound were picked for the MRM settings. The peak areas of ion currents versus five concentrations (0.3125, 0.625, 1.25, 2.5, and 5 ppm) of the standard compounds were used to construct the calibration curves. By the developed quantitative protocols, the four compounds (1-4) were analyzed to be 1.23%, 3.32%, 0.56%, and 0.89%, respectively. Taking into consideration the IC 50 value of LJEE (10.16 µg/mL) on superoxide anion inhibition, the total content of the sum of the three major chlorogenic acids, 5.11%, contributed a reasonable inhibitory activity (IC 50 values: 1.33-1.40 µg/mL), indicating that the anti-inflammatory potential of LJEE on activated neutrophils is mainly attributed to its content of chlorogenic acid derivatives. Apart from the chlorogenic acid derivatives, flavonoids/flavonoids glycosides from LJ, such as luteolin, ochnaflavon, luteolin-7-O-β-D-glucopyranoside, and quercetin 3-Oβ-D-glucopyranoside, were also reported to exhibit anti-inflammatory and antioxidative properties [47][48][49]. Luteolin effectively attenuated the lipopolysaccharide (LPS)-induced tumor necrosis factor-α, interleukin-6, and inducible nitric oxide production in vitro, as well as protected against LPS-induced lethal toxicity by inhibiting pro-inflammatory molecule expression in vivo [47]. Ochnaflavon was found to significantly decrease cyclooxygenase-2 (COX-2)-dependent prostaglandin D2 (PGD2) generation in mast cells, exhibiting dual COX-2/5-1ipoxygenase inhibitory properties [49]. Both luteolin-7-O-β-D-glucopyranoside and quercetin 3-O-β-D-glucopyranoside were shown to be potent antioxidants in the DPPH scavenging assay [48]. Moreover, in the septic mouse model, an ethanolic crude extract of LJ, HS-23, was found to alleviate septic injury by inhibiting toll-like receptor 4 signaling, evidenced by the downregulation in protein expressions of myeloid differentiation primary response protein 88, p38 and c-Jun N-terminal kinase, TIR-domain-containing adapterinducing interferon-β, and interferon regulatory transcription factor 3 [50]. Therefore, it is suggested that the anti-inflammatory and antioxidative activities of these ethanolsoluble flavonoid derivatives and crude extracts may also support the use of LJEE in heat-clearing applications.

Lonicerae Japonicae Flos Water Extract (LJWE) Extracts Interrupted the Binding of SARS-CoV-2 Spike/ACE2
The receptor-binding domain (RBD) of SARS-CoV-2 coronavirus spike proteins plays the role of a critical determiner of viral tropism and infectivity [51]. The current study applied an HTRF SARS-CoV-2 spike/ACE2 binding assay kit to determine if the binding levels between the SARS-CoV-2 spike protein and ACE2 protein were affected by LJ extracts. The results indicated that the three LJ extracts (LJWE, LJEE, and LJME), as well as the LJWE (IC 50 : 27.52 ± 1.26 µg/mL), showed a suppressive effect on reducing the SARS-CoV-2 spike/ACE2 binding ( Figure 7A,B). This data revealed the interference effect of LJWE on SARS-CoV-2 infection.
activities of these ethanol-soluble flavonoid derivatives and crude extracts may also sup-port the use of LJEE in heat-clearing applications.

Lonicerae Japonicae Flos Water Extract (LJWE) Extracts Interrupted the Binding of SARS-CoV-2 Spike/ACE2
The receptor-binding domain (RBD) of SARS-CoV-2 coronavirus spike proteins plays the role of a critical determiner of viral tropism and infectivity [51]. The current study applied an HTRF SARS-CoV-2 spike/ACE2 binding assay kit to determine if the binding levels between the SARS-CoV-2 spike protein and ACE2 protein were affected by LJ extracts. The results indicated that the three LJ extracts (LJWE, LJEE, and LJME), as well as the LJWE (IC50: 27.52 ± 1.26 μg/mL), showed a suppressive effect on reducing the SARS-CoV-2 spike/ACE2 binding ( Figure 7A,B). This data revealed the interference effect of LJWE on SARS-CoV-2 infection.

Conclusions
The anti-inflammatory effect and underlying pharmacological mechanisms of Lonicerae japonicae flos (LJ) on activated human neutrophilic inflammation were illustrated for the first time. The chlorogenic acid-enriched LJEE successfully attenuated inflammatory reactions in the activated neutrophils, including superoxide anion generation, release of elastase, CD11b expression, chemotactic migration, cell adhesion, and NET formation. Calcium mobilization played a crucial role in the anti-neutrophilic inflammatory mechanism of action of LJEE. The tandem mass-based qualitative and quantitative analyzing protocols of the phytoconstituents from LJEE were established. Moreover, LJWE was found to interfere with the infecting process of SARS-CoV-2. Since both neutrophilic inflammation and SARS-CoV-2/ACE2 bindings are crucial for COVID-19 infection, the daily use of LJ during the COVID-19 pandemic is suggested.

Conclusions
The anti-inflammatory effect and underlying pharmacological mechanisms of Lonicerae japonicae flos (LJ) on activated human neutrophilic inflammation were illustrated for the first time. The chlorogenic acid-enriched LJEE successfully attenuated inflammatory reactions in the activated neutrophils, including superoxide anion generation, release of elastase, CD11b expression, chemotactic migration, cell adhesion, and NET formation. Calcium mobilization played a crucial role in the anti-neutrophilic inflammatory mechanism of action of LJEE. The tandem mass-based qualitative and quantitative analyzing protocols of the phytoconstituents from LJEE were established. Moreover, LJWE was found to interfere with the infecting process of SARS-CoV-2. Since both neutrophilic inflammation and SARS-CoV-2/ACE2 bindings are crucial for COVID-19 infection, the daily use of LJ during the COVID-19 pandemic is suggested.  Institutional Review Board Statement: The study was conducted in accordance with the Declaration of Helsinki and was approved by the Institutional Review Board (IRB) at Chang Gung Memorial Hospital (protocol code No. 202002493A3, approval date: 6 February 2020) for studies involving humans.