Antioxidant and Anti-Inflammatory Capacities of Fractions and Constituents from Vicia tetrasperma

The young leaves and shoots of V. tetrasperma are consumed daily as cooked vegetables and can provide various health benefits. The antioxidant and anti-inflammatory capacities of its total extract and fractions were accessed for the first time in this study. The bioactivities guided the separation of the active fraction (EtOAc), leading to the identification of nine flavonoid glycoside compositions from this plant for the first time. In addition, the fractions and all isolates were evaluated for their inhibition against NO and IL-8 production in LPS-stimulated RAW264.7 and HT-29 cell lines, respectively. The most active ingredient was further assayed for its inhibitory abilities to iNOS and COX-2 proteins. Indeed, its mechanisms of action modes were confirmed by Western blotting assays through the reduction in their expression levels. An in silico approach revealed the substantial binding energies of docked compounds into established complexes to verify their anti-inflammatory properties. In addition, the presence of active components in the plant was validated by an established method on the UPLC-DAD system. Our research has boosted the value of this vegetable’s daily use and provided a therapeutic approach for the development of functional food products for health improvement regarding the treatment of oxidation and inflammation.


Introduction
Antioxidants and inflammatory inhibitors are served as therapeutic interventions for the treatment of disease development. For example, antioxidant therapy could delay the progression of atherosclerotic lesions [1]. Antioxidants are also able to regress pre-malignant lesions or inhibit them from developing into cancer by interfering with the metabolic activation of chemical carcinogens [2]. On the other hand, excessive levels of ROS cause protein and DNA damage, leading to inflammation or mutation [3]. In the same manner, inflammation releases many mediators and cytokine production. This progression directly results in the body's cells and tissue injuries [4]. Therefore, anti-inflammatory inhibitors have been developed and widely used for inflammatory diseases, such as etanercept and toculizumab, for the treatment of rheumatoid arthritis by reducing TNF-α and IL-6 activities, respectively [5,6]. However, some current anti-inflammatory inhibitors may cause some adverse effects such as glucocorticoids [7]. Thus, it is necessary to find active and safe antioxidant and anti-inflammatory inhibitors for the treatment of diseases.

UPLC-ESI-QTOF-MS Assay
The mass fragmentation of isolated compounds (1-9) was performed on a Waters UPLC system (Waters, Milford, MA, USA) coupled with Waters column and a Xevo G2-XS Q-TOF MS with electrospray ionization source (Waters, Milford, MA, USA). Triple TOF MS equipped with a DuosprayTM ion source was used to complete the high-resolution experiment. For mass detection, the instrument was operated in the negative ion electrospray mode, and the conditions of the MS/MS detector were as follows: the de-solvation gas was 800 L/h at 400 • C, the cone gas was 50 L/h, the source offset voltage was 80 eV, and the source temperature was 120 • C. A full scan was run in the negative mode with a mass range from m/z 50 to 1250 amu. The capillary voltage was 3.0 kV, and the sampling cone voltage was 40 eV. Sodium formate was used for mass spectrometer instrument calibration in the resolution mode. Leucine encephalin, which generated the reference ion (m/z 554.2615 [M-H]-), was used to ensure accuracy throughout the mass spectrometry analysis. All data acquisition and processes were performed for qualitative analysis by using MassLynx V4.1 and UNIFI V1.9 (Waters, Milford, MA, USA). The gradient elution of the mobile phase was conducted following the above established gradient elution.

Cell Culture and Cell Viability
Mouse macrophages (RAW264.7) and human colon epithelial (HT-29) cells were acquired from the Korean Cell Lines Bank (Seoul, Republic of Korea). These cell lines were grown at 37 • C and were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated FBS, streptomycin sulfate (100 µg/mL), and penicillin (100 IU/mL) in a humidified atmosphere of 5% CO 2 . RAW264.7 cells were seeded into 96-well plates followed by preincubation for 24 h before addition with samples for 1 h, and then stimulation with LPS (1 µg/mL) for 16 h, whereas HT-29 cells were seeded into 96-well plates, preincubated for 24 h, then treated with 100 µM of compounds (1-9) for 2 h, and stimulated with LPS (100 ng/mL) for 12 h. The cultured cells were incubated with MTT (5 mg/mL) at 37 • C for four hours. The supernatants were removed and 100 µL dimethyl sulfoxide (DMSO) (SigmaAldrich, Saint Louis, MO, USA) was added. After five minutes, the absorbance of the formazan crystals was measured at 570 nm in a microplate reader (Bio Tek Instruments, Winooski, VT, USA).

Measurement of ROS Accumulation
RAW264.7 were seeded in a 6-well plate at concentration of 1 × 10 4 cells/well for 20 h and then treated with positive control (ascorbic acid, A.A) and samples (total extract and fractions) at a concentration of 100 µg/mL. Subsequently, 10 µM dichlorodihydrofluorescein diacetate (DCFH-DA) diluted with 1/1000 in a serum-free medium was added after removing all the cell culture medium. After incubation for 20 min at 37 • C in dark, the cells were washed 3 times with PBS to remove DCFH-DA, which had not been entered into cells. The cells were visualized by an inverted fluorescence microscope and further detected by a fluorescent microplate reader at 485 and 530 nm.

Measurement of NO Production
The level of NO production was determined by measuring the amount of secreted nitrite from the cell culture supernatants, as described previously [20]. Briefly, RAW264.7 cells (1 × 10 5 cells/well) were pretreated with samples (total extract, fractions, 100 µg/mL, and isolates 1-9, 100 µM) for 1 h and stimulated with LPS (1 µg/mL) for 20 h. Then, the collected supernatant was incubated with an equal volume of Griess reagent by using equal volumes of 1% (w/v) sulfanilamide in 5% (v/v) phosphoric acid and 0.1% (w/v) N-(1-naphtyl) ethylenediamine at room temperature for 10 min. The absorption was measured at 550 nm by microplate reader (Bio-Tek Instruments, Inc., Winooski, VT, USA). The LPS-induced NO production was measured in the treated samples and controls (with and without LPS in the absence of sample addition).

DPPH and ABTS Assay
The scavenging abilities of samples on DPPH and ABTS radicals were assayed following a previous method [20] in which total extract and fractions were prepared at concentration of 100 µg/mL for the experiments. Trolox was considered a reference standard. The calibration curve was built using various concentrations of Trolox (0-100 µM). Results were presented as µmol of Trolox equivalent per gram of extract, and were compared to the Trolox standard curve.

Western Blotting
The reduction ability of iNOS and COX-2 protein expression on RAW264.7 cells of the strong Active Compound 6 and total extract was examined, following our previous study [21]. Briefly, RAW264.7 cells were seeded and cultured using the above-explained experimental conditions in 6-well plates for 24 h. Then, the cultured cells were treated with Compound 6 and total extract at concentrations of 100, 200 µM and 100, 200 µg/mL, respectively, for 1 h, then stimulated with LPS (1 µg/mL). After 24 h, (iNOS and COX-2) cells were washed with cold phosphate-buffered saline (PBS) twice. The cell lysates were extracted using a protein extraction solution (proprep, iNtRON, Biotechnology, Daejeon, Korea). The protein concentration was determined by the Bradford assay method. Equal amounts of control, LPS, and compound-treated samples (30 µg) were separated by 10% SDS-PAGE gels and transferred on PVDF membranes. The membranes were blocked with 5% skim milk powder in plain buffer [20 mM Tris-HCl (pH 7.4) and 4 M NaCl] for one hour at room temperature. The membranes were incubated with primary antibodies (iNOS, COX-2, and β-actin) at 4 • C overnight, and then washed three times with a wash buffer [1 M Tris-HCl (Ph 7.4), 4 M NaCl, Tween-20 in DW] for 10 min. They were incubated in specific secondary antibodies (1:2000 dilution) conjugated with horseradish peroxidase at RT for two hours and washed. The protein signals were obtained using chemiluminescence detection reagents (Thermo Fisher Scientifc, Waltham, MA, USA) and imaged using a bio-imaging system (MicroChemi 4.2 Chemilumineszenz-System, Modi'in-Maccabim-Re'ut, Israel).

Measurement of IL-8 Production
The IL-8 production assay was used on HT-29 cells (3 × 10 5 cells/well) in 96-well plates using ELISA kit (BD OptEIATM, CA, USA) following the manufacturer's instructions.

Molecular Docking
The 3D structures of iNOS (PDB ID: 3E7G), COX-2 (PDB ID: 5IKQ), and IL-8 (PDB ID: 5D14) proteins were obtained from the RCSB protein data bank (https://www.rcsb.org; accessed on 12 October 2022). Proteins and ligand were prepared using MGL tools 1.5.6. The structures of receptors were processed by removing water, adding polar hydrogen atoms, and Kollman charges. Structure (6 -acetylapiin, 6) was downloaded from PubChem (https: //pubchem.ncbi.nlm.nih.gov, 12 October 2022) in sdf formats, and structures (apigenin- 9) were prepared using by Avogadro package via force field method-MMFF94. Then, the geometries of these structures were transformed into pdbqt format using the Open Babel. The ligand conformations were performed by adding Gasteiger charges. A grid box of coordinates was determined using Pymol. A total of 100 runs were conducted under default parameters using the Lamarckian genetic algorithm. The protein-ligands docking calculations were performed using AutoDock 4.2. Residues-ligand interactions were visualized with Discovery studio 2021.

UPLC-DAD System and Separation Condition
The sample was analyzed using a Waters UPLC system (Waters, Milford, MA, USA) equipped with an autosampler, column temperature controller, and DAD detector. A Waters Acquity UPLC HSS T3 column (2.1 × 100 mm, 1.8 µm) was used to carry out the experiment at 40 • C. The mobile phases were buffered with 0.1% formic acid for both aqueous (water, A) and organic (acetonitrile, B) elements. The flow rate was set to 0.4 mL/min, and the injection volume was 2 µL. The gradient elution of the mobile phase was conducted as follows: 0-1 min (B: 5 %), 1-4 min (B: 5-15%), 4-12 min (B: 15-35%), 12-17 min (B: 35-45%), 17-23 min (B: 45-100%). Then, it was held at 100% B for 3 min and then returned to the initial conditions for re-equilibration. The range of DAD detection wavelength was set to 200-400 nm and chromatograms were recorded at 254 nm. Preparation of standard solutions: a standard mixture including analytes was dissolved in 100% methanol as a stock solution and then diluted with 100% methanol to obtain working concentrations for calibration curves. The solution was sealed by an elastic-plastic film and stored in the refrigerator at 4 • C for analysis.

Preparation of Calibration Standard Solution
Sample preparation: a dried herb of V. teterasperma (1 g) was extracted with 100% ethanol (10 mL) by sonication for 90 min. The extracted solution was filtered through filter paper. Its solvent was removed at room temperature using an evaporator under nitrogen gas. For a quantitative analysis of the V. terterasperma, a total extract (13 mg) was dissolved in 1 mL of methanol and sonicated for 5 min. The sample solutions were then filtered through a 0.22 µm PTFE syringe filter and the filtrates were collected and stored at 4 • C prior to use.
Linearity, LODs, LOQs: the identification of standards in the sample was accessed by calculating relative retention time and UV absorption maxima in comparison to those of standard mixture at the same UPLC analytical conditions. The UPLC-DAD method was applied to conduct the experiment with validation parameters, including linearity, limit of detection (LOD), limit of quantification (LOQ), precision, and accuracy, in accordance with the guidelines described at the international conference on Harmonisation, whereas the calibration curves were built using the regression equation based on the peak areas (y) in response to the corresponding concentrations (x, µg/mL) of six markers in the standard solutions at eight different concentrations, and the correlation coefficients (r 2 ) were calculated. The LOD values were calculated using LOD = 3.3 × SD/S. In addition, the LOQ values were calculated using LOQ = 10 × SD/S, while SD was the standard deviation and S was the slope of the calibration curve.
Intra-and inter-day precision were performed six times during the same day and on six successive days, respectively. The relative standard deviation (RSD) was obtained using the following equation: RSD (%) = SD × 100/mean measured concentration.
To prove the repeatability of the analytical method, the accuracy was experimented with by spiking the known amounts of the standards into a working solution. Their concentrations were prepared at three (high, medium, and low) levels such as Compounds 1 and 3 (250, 100, 62.5 ug/mL), Compounds 4 and 5 (60, 24, 15 ug/mL), and Compounds 6 (200, 50, 31.5 ug/mL) and 7 (20, 8, 5 ug/mL). Then, a recovery test was performed using the above analytical condition. The recovery (%) was calculated using the following equation:

Statistical Analysis
Data were presented as mean ± SD (n = 3) of at least three replicates. The nonparametric one-way ANOVA followed by Turkey's multiple comparison test using the Graphprism version 8.0.1 software (Graphpad Software, La Jolla, CA, USA) was used for statistical analyses. * p < 0.05, ** p < 0.01, compared to controls, were considered significant values.

Antioxidant and Anti-Inflammatory Capacities of Total Extract and Fractions
The total extract of V. tetrasperma was screened for its antioxidant effect using an ROS assay [22] with some modifications by using RAW264.7 cells. The total extract of V. tetrasperma exhibited the significant inhibition of ROS production in LPS-mediated RAW264.7 cells with an inhibition rate of 52.1% at a concentration of 100 µg/mL, compared to the positive control (ascorbic acid, 76.9%), without cytotoxic effects (cell viability > 90%). Thus, the total extract was suspended in water and partitioned into n-hexane, CH 2 Cl 2 , EtOAc, n-BuOH, and distilled water (DW) fractions. The cell viability of the total extract and fractions were conducted on RAW264.7 cells. The result revealed that the n-hexane fraction showed some toxic effects on cells. Thus, this fraction was eliminated for the ROS experiment. Subsequently, CH 2 Cl 2 , EtOAc, n-BuOH, and DW fractions were examined for their antioxidant effect using an ROS assay. Among the tested samples, EtOAc and MC fractions showed potential for antioxidant effects with inhibition rates at 95.4% and 91.8%, respectively, stronger than those of positive control (ascorbic acid, 76.9%) at the tested concentration of 100 µg/mL. n-BuOH and DW fractions showed weak antioxidant effects (Figure 1). The abilities of the total extract and fractions were also evaluated for their anti-inflammatory effect against NO production. The EtOAc and CH 2 Cl 2 fractions showed strong inhibition of NO production with suppressions of 45.5 and 45.9%, respectively, at 100 µg/mL. The n-BuOH and DW fractions displayed weaker inhibitory effects with inhibition rates of 30.5 and 22.5% (compared to those of the LPS-treated group), respectively, at 100 µg/mL. Additionally, the EtOAc showed the highest antioxidant capacities among all the fractions for both radical scavenging activities on DPPH • and ABTS •+ (Table 1). Therefore, the strong active fraction (EtOAc) was selected as the material for separation. pared to the positive control (ascorbic acid, 76.9%), without cytotoxic effects > 90%). Thus, the total extract was suspended in water and partitioned i CH2Cl2, EtOAc, n-BuOH, and distilled water (DW) fractions. The cell viabil extract and fractions were conducted on RAW264.7 cells. The result reveal hexane fraction showed some toxic effects on cells. Thus, this fraction was the ROS experiment. Subsequently, CH2Cl2, EtOAc, n-BuOH, and DW frac amined for their antioxidant effect using an ROS assay. Among the tested sa and MC fractions showed potential for antioxidant effects with inhibition and 91.8%, respectively, stronger than those of positive control (ascorbic a the tested concentration of 100 μg/mL. n-BuOH and DW fractions showed we effects (Figure 1). The abilities of the total extract and fractions were also eval anti-inflammatory effect against NO production. The EtOAc and CH2Cl2 frac strong inhibition of NO production with suppressions of 45.5 and 45.9%, re 100 μg/mL. The n-BuOH and DW fractions displayed weaker inhibitory eff bition rates of 30.5 and 22.5% (compared to those of the LPS-treated group) at 100 μg/mL. Additionally, the EtOAc showed the highest antioxidant cap all the fractions for both radical scavenging activities on DPPH • and ABT Therefore, the strong active fraction (EtOAc) was selected as the material fo

Identification of Constituents
The most active fraction (EtOAc), which has potent antioxidant and anti-inflammatory capacities, was separated using multiple chromatographic methods to yield nine compounds (1-9) (Figure 2; Figures S1-S32).

Anti-Inflammatory and Cytotoxic Capacities
To find the beneficial compounds that have anti-inflammatory properties, the inhibition of NO production was examined in LPS-stimulated RAW264.7 cells by the isolated compounds (1-9). At the tested concentration of 100 µM, Compounds 3, 4, and 6 strongly reduced NO production by 72.7, 71.6 and 74.8% compared to the LPS-treated control, respectively, and Compounds 1, 2, 5, and 7-9 displayed a significant inhibition of LPS-induced NO production by 66.7 to 53.5% at the same tested concentration. None of these isolates were cytotoxic ( Figure 5).

Anti-Inflammatory and Cytotoxic Capacities
To find the beneficial compounds that have anti-inflammatory properties, the inhibition of NO production was examined in LPS-stimulated RAW264.7 cells by the isolated compounds (1-9). At the tested concentration of 100 μM, Compounds 3, 4, and 6 strongly reduced NO production by 72.7, 71.6 and 74.8% compared to the LPS-treated control, respectively, and Compounds 1, 2, 5, and 7-9 displayed a significant inhibition of LPS-induced NO production by 66.7 to 53.5% at the same tested concentration. None of these isolates were cytotoxic ( Figure 5).

Inhibitory Mechanism of Flavonoid Glycosides against iNOS and COX-2 Levels Expression
The action modes of the most active compound (6) were further examined for both enzymatic proteins. A Western blotting assay was, therefore, conducted to elucidate the potential mechanism. The inhibition of the iNOS and COX-2 expressions by the strongest active compound (6) and the total extract were investigated ( Figure 6). Compound 6 and the total extract markedly reduced the expression levels of both inflammatory genes in

Inhibitory Mechanism of Flavonoid Glycosides against iNOS and COX-2 Levels Expression
The action modes of the most active compound (6) were further examined for both enzymatic proteins. A Western blotting assay was, therefore, conducted to elucidate the potential mechanism. The inhibition of the iNOS and COX-2 expressions by the strongest active compound (6) and the total extract were investigated ( Figure 6). Compound 6 and the total extract markedly reduced the expression levels of both inflammatory genes in RAW264.7 cells compared to those of β-actin in LPS-pre-treated cells without added sample (Figure 7). The result suggested that these inhibitors may affect NO production by inhibiting catalyst enzymes iNOS and COX-2 in the rate-limiting steps to produce NO and PGE2, respectively.
Antioxidants 2023, 12, x FOR PEER REVIEW 13 of 20 RAW264.7 cells compared to those of β-actin in LPS-pre-treated cells without added sample (Figure 7). The result suggested that these inhibitors may affect NO production by inhibiting catalyst enzymes iNOS and COX-2 in the rate-limiting steps to produce NO and PGE2, respectively.   Antioxidants 2023, 12, x FOR PEER REVIEW 13 of 20 RAW264.7 cells compared to those of β-actin in LPS-pre-treated cells without added sample ( Figure 7). The result suggested that these inhibitors may affect NO production by inhibiting catalyst enzymes iNOS and COX-2 in the rate-limiting steps to produce NO and PGE2, respectively.

Anti-Inflammatory Capacity on IL-8 Production in LPS-Stimulated HT-29 Cells
In addition, all the isolates (1-9) were evaluated for their ability to inhibit IL-8 production. Compared to the LPS-treated control without sample addition, Compound 9 exhibited the strongest activity of LPS-induced IL-8 production in HT-29 cells, with an inhibition rate of 77.4% at 100 µM. Compound 8 showed a significant inhibition rate of 70.9%, followed by other compounds (1-7) with inhibition rates ranging from 57.3 to 52.1% compared to LPS-induced IL-8 production without sample pre-treatment at the same tested concentration of 100 µM. In contrast, no tested samples showed any significant cytotoxicity in HT-29 cells at the tested conditions by the MTT assay (Figure 7).

Molecular Docking Analysis
Molecular docking simulation was applied to compute the binding affinity and interactions between receptors and Compounds 6, 8, and 9 in silico discovery ( Figures S33-S35). The result showed that the iNOS-Compound 6 complex exhibited a strong docking energy of −9.50 kcal/mol. This may be explained by the interactions between Compound 6 and the active residues of iNOS protein ( Figure 8, Table 3).

Anti-Inflammatory Capacity on IL-8 Production in LPS-Stimulated HT-29 Cells
In addition, all the isolates (1-9) were evaluated for their ability to inhibit I duction. Compared to the LPS-treated control without sample addition, Compou hibited the strongest activity of LPS-induced IL-8 production in HT-29 cells, with bition rate of 77.4% at 100 μM. Compound 8 showed a significant inhibition rate o followed by other compounds (1-7) with inhibition rates ranging from 57.3 to 52. pared to LPS-induced IL-8 production without sample pre-treatment at the sam concentration of 100 μM. In contrast, no tested samples showed any significant city in HT-29 cells at the tested conditions by the MTT assay (Figure 7).

Molecular Docking Analysis
Molecular docking simulation was applied to compute the binding affinity teractions between receptors and Compounds 6, 8, and 9 in silico discovery ( Figu  S35). The result showed that the iNOS-Compound 6 complex exhibited a strong energy of −9.50 kcal/mol. This may be explained by the interactions between Co 6 and the active residues of iNOS protein ( Figure 8, Table 3).   In detail, the ligand was stabilized by the formation of 7 conventional hydrogen bonds with amino acids, including ARG199, ARG381, ASP382, ILE201, GLU377, GLY202, and TRO463 along with a hydrophobic interaction at VAL352. The above observation suggested that the docked ligand occupied the binding pocket of the iNOS structure. Moreover, the iNOS-compound complex showed additional interactions (Table 2) with active residues in the binding pocket of the iNOS protein ( Figure 9) in which the amino acids GLU377, ARG381, and VAL352 are the important residues of the binding sites in the pocket [30][31][32].   In detail, the ligand was stabilized by the formation of 7 conventional hydrogen bonds with amino acids, including ARG199, ARG381, ASP382, ILE201, GLU377, GLY202, and TRO463 along with a hydrophobic interaction at VAL352. The above observation suggested that the docked ligand occupied the binding pocket of the iNOS structure. Moreover, the iNOS-compound complex showed additional interactions ( Table 2) with active residues in the binding pocket of the iNOS protein ( Figure 9) in which the amino acids GLU377, ARG381, and VAL352 are the important residues of the binding sites in the pocket [30][31][32].  Furthermore, the COX-2-Compound 6 docked complex showed docking energy of −3.28 kcal/mol (Table 3) along with the formation of three hydrogen bonds at ASP157, GLN457, and HIS214 ( Figure 8). Moreover, all the ligands exhibited additional interactions including hydrophobic, van der Waals, and polar interactions with active residues surrounding them in the binding pocket of the protein. Table 3, Compound 9 displayed a lower binding affinity (−7.75 kcal/mol) than that (−4.32 kcal/mol) of Compound 8 when it was docked to the 5D14 (IL-8) receptor. This observation is associated with their strong inhibition on IL-8 production as seen in the above experimental data. Importantly, these ligands showed interactions with amino acids GLN6, LYS9, and CYS48 surrounding Compound 8 and those of ARG4 and CYS48 bounding Compound 9 through conventional hydrogen bonds, respectively, at the binding pockets of established complexes. On the other hand, these ligands also displayed interactions with other active residues of the IL-8 protein.

As shown in
Thus, these docking analyses support the data approach of the above Western blotting and IL-8 experimental results.

Establishment and Validation Analysis of Marker Compounds (1 and 3-7) from the Herbal Extract of V. teterasperma
An analytical method was established and applied to build a chromatogram of the V. teterasperma extract. The chromatographic profile was obtained by optimizing the analytical factors, including the mobile phase, gradient elution, column, wavelength detection, and flow rate, as well as resolution for peak separation (Figure 9).
Linearity regression, LODs, and LOQs: linearity is the capability to obtain data that are proportional to the concentration of the analyte in the sample within a given range. It was calculated between peak areas and concentrations, and was performed using six calibration curves built at different concentrations of the standard solutions of each analyte, with the correlation coefficients (r 2 ) ranging from 0.9990 to 0.9997. These data indicated that the responses of the standards in the working ranges of the concentrations were linear and satisfied. The LOD and LOQ values, which expressed the sensitivity of the system, were calculated, and ranged from 0.12 to 0.60 µg/mL and from 0.39 to 1.84 µg/mL, respectively (Table 4).

Qualification of Marker Compounds from the V. tetrasperma Extract
The analytical method aimed to qualify the content of an individual marker compound in the total extract of the plant. The above established method was applied to determine the average content of each marker compound in the total extract of V. tetrasperma. Compound 3 had the highest content of 3.02 mg/g. Compounds 1 and 5 displayed medium amounts of 2.18 and 1.94 mg/g, respectively. Compound 4 had a content of 0.39 mg/g. Compounds 6 and 7 exhibited similar contents at 0.09 and 0.10 mg/g, respectively (Table 4).

Discussion
The antioxidant and anti-inflammatory effects of the total extract and fractions derived from V. teterasperma guided the separation and identification of nine compounds from the active (EtOAc) fraction. The structures of isolated compounds (1-9) were established by using spectroscopic techniques and compared to those reported in the literatures.
was then employed to quantitate the marker content from the extract. As a result, the active compounds, Compounds 1 and 3, expressed high levels with amounts of 2.18 and 3.02 mg/g, respectively, reasonably considered as important components promoting the anti-inflammatory capacity of the extract of this plant. On the other hand, the content of other active components, Compounds 4 and 6, with amounts of 0.39 and 0.09 mg/g, respectively, may contribute to the enhancement of the anti-inflammatory property of this plant.
Further studies should be performed with a large amount of dried material to enhance the study of the biomass of active compounds found in this primary study which provides support for in vivo studies in the future.

Conclusions
In this study, we firstly reported antioxidant and anti-inflammatory activities through DPPH/ABTS radical scavenging activities, as well as inhibitory effects against ROS, and NO production, respectively. The current study also revealed the separation of flavonoid glycosides obtained from V. tetrasperma for the first time, which resulted in the identification of nine compounds. Among them, chrysoeriol 7-O-(2-apiosyl-6-acetyl)-glucoside was reported for the first time in NMR data. This is also the first report on the antioxidant and anti-inflammatory properties of this herbal food by inhibiting NO and IL-8 production in RAW264.7 and HT-29 cells, respectively. We firstly reported on the inhibitory effects of Compounds 1 and 4-7 in LPS-induced NO production on RAW264.7 cells and in LPSinduced IL-8 production on HT-29 cells, respectively. Further experiments with the strongly active compound were conducted to assess the inhibitory mechanism of suppressing (iNOS and COX-2) isoform expression for the first time. The active compounds with antiinflammatory effects and their content in the herbal extract clarified the inhibitory capacity of V. tetrasperma. The molecular docking analysis further proved the anti-inflammatory mode of action by inhibiting the enzymatic activities of iNOS, COX-2, and of the IL-8 protein during the reaction process to produce mediators and cytokine production. The interesting activities this study has found indicate the new value of V. tetrasperma, which is consumed as a vegetable daily, and offered some evidence for the usage and development of this plant in the field of functional food to guide the treatment of inflammatory diseases in the future. This study also provided an effective tool for assessing the quality of this herbal food on the market through a UPLC analytical method using the above six standards. Our findings highlight the significance of this herb as a potential source of previously unrecognized health-promoting substances.

Conflicts of Interest:
The authors declare no conflict of interest.