The Cactus (Opuntia ficus-indica) Cladodes and Callus Extracts: A Study Combined with LC-MS Metabolic Profiling, In-Silico, and In-Vitro Analyses

Opuntia ficus-indica (OF) phytochemicals have received considerable attention because of their health benefits. However, the structure-activity relationship between saponin and flavonoid antioxidant compounds among secondary metabolites has rarely been reported. In a molecular docking study, selected compounds from both Opuntia ficus-indica callus (OFC) and OF ethanol extract were found to be involved in Toll-like receptor 4 and mitogen-activated protein kinase (MAPK) signaling pathways. High affinity was specific for MAPK, and it was proposed to inhibit the oxidative and inflammatory responses with poricoic acid H (−8.3 Kcal/mol) and rutin (−9.0 Kcal/mol). The pro-inflammatory cytokine factors at a concentration of 200 μg/mL were LPS-stimulated TNF-α (OFC 72.33 ng/mL, OF 66.78 ng/mL) and IL-1β (OFC 49.10 pg/mL, OF 34.45 pg/mL), both of which significantly decreased OF (p < 0.01, p < 0.001). Taken together, increased NO, PGE2, and pro-inflammatory cytokines were significantly decreased in a dose-dependent manner in cells pretreated with OFC and the OF extract (p < 0.05). These findings suggest that OFC and OF have important potential as natural antioxidant, anti-inflammatory agents in health-promoting foods and medicine.


Introduction
Today's population is approximately 7.96 billion people, and about 50% of the population is concentrated in warmer regions (20-60 • north latitude). Modern human ecology is polarized according to the geography of the earth due to radical industrialization since the 1960s [1]. This unbalanced population distribution is facing a singularity in the ecological environment. In addition, environmental changes caused by global warming will affect land agriculture in the future by causing phenomena such as land flooding due to sea level rises and increased frequency and intensity of heat waves due to surface temperature rises. Environmentally friendly soilless cultivation technology is a promising approach for these problems of future agriculture [2].
Plant cell culture is a cultivation technology that can aseptically culture plant cells and organs using a medium containing nutrients, and it can uniformly mass-produce throughout the year without soil [3]. Populations of single cells, regenerating into fully functional plants, are grown in containers filled with a variety of nutrient-rich growing media [4]. Cultivation methods can improve environmental control and avoid uncertainties in soil water and nutrient status. Callus cultures are plant tissues of improved quality that are formed as meristems masses derived from plant wounds or plant cells. These transgenic plants can be used to confirm the function of a specific gene or to cultivate new crop varieties with agriculturally useful traits, and callus cultures extracts have been in the spotlight as useful materials that can be used for cosmetics or food.
Two species of Opuntia are used as food in Korea (O. ficus-indica and O. humifusa). O. ficus-indica is a naturalized cactus on Jeju Island, Republic of Korea (originating from Mexico), and its fruit is called nopal (baeknyeoncho in Korean). O. humifusa grows in Gyeonggi-do, Republic of Korea. The Korean names are opuntieae, baeknyeoncho, bogum, and palm, which were coined in various forms influenced by lifestyles. These cacti are used as food after processing them into dried powder, extract, or concentrate or after fermentation, and the fruit is used for oil and beverages. Cacti are plants specialized for a dry environment. They adapt according to the influence of extreme natural environmental factors (light, seasonal change, temperature, moisture, disease, insects, soil, etc.). In addition, substances useful to humans (phytochemicals) have been obtained. Despite being a potential nutraceutical, the parts used (cladode and fruit) are limited.
As an important reservoir of various biologically active compounds, called phytochemicals, plants are essential for treating several human diseases [5]. Phytochemicals are chemical products, including defense factors, that are metabolized by all plants when they are exposed to various environmental factors (ultraviolet rays, pests, microorganisms, etc.) from seed to adult growth [6]. Growing plants can synthesize and accumulate a comprehensive spectrum of chemical compounds in response to physiological stimuli and stress [7]. Representative components of phytochemicals include saponin, phenolic acid, flavonoids, and carotenoids. Plants with these functional components are used for food and medicine and have been highlighted as useful raw materials for healing various defects in the human body.
In this study, a molecular docking study was performed to determine whether cactus and callus extracts could competitively bind to Toll-like receptor 4 and mitogen-activated protein kinase and block lipopolysaccharide-induced inflammation. Anti-inflammatory activity and phytochemical components of cactus and callus were identified and observed.

Plant Materials and Reagents
Plant materials: Opuntia ficus-indica (OF) plant material, i.e., mature cladodes as a dried powder, was purchased from Jeju Island of Republic of Korea (33 • 22 30.5 N 126 • 12 57.0 E, m.jeju100.com (accessed on 19 May 2020), 5 years old). To induce Opuntia ficus-indica callus (OFC) growth, OF was first disinfected in a 5 mM salicylic acid solution for 2 min, 70% ethanol for 10 min, and 1% bleach (sodium hypochlorite) for 10 min and then was washed three times in sterile distilled water for 15 min. Sterilized OF was cut into 1 × 1 cm 2 pieces and inoculated into media. For media preparation, agar (8 g/L), sucrose (10.09 g/L), and 3-(N-morpholino) propane sulfonic acid (MOPS; MB Cell Company, Seoul, Republic of Korea) (0.5 g/L) were added to 2,4-dichlorophenoxy acetic acid medium (2,4-D medium; MB cell Company, Seoul, Republic of Korea) (1 mg/L). The media were adjusted to a pH of 5.7 and autoclaved at 121 • C for 15 min before use. Sterilized OF was cultivated in media at room temperature for about 4 weeks. Calluses were then harvested and cultured with liquid medium without agar at room temperature for 2 weeks. OFC extracts were then filtered, vacuum-concentrated, and freeze-dried in the experiments described below. Samples were stored at −70 • C for further experimental work.

Sample Preparation
The weighed OF and OFC dried powder (1.0 g) were suspended in 10 mL of 70% ethanol containing the extract on a bullet blender tissue homogenizer (Next Advance, NY, USA). After centrifugation (3000 rpm, 4 • C, 15 min), the supernatant was immediately filtered using a syringe filter (PVDF 0.20 µm, Whatman, Kent, United Kingdom), and then 0.5 mL of the filtrate was diluted with water to a final volume of 5 mL. The experiments were performed in triplicate.

UPLC-QTOF-MS Metabolic Profiling
These samples were analyzed using Waters Acquity UPLC-QTOF. The sample extract was injected into an Acquity UPLC BEH C18 column (2.1 mm × 100 mm, 1.7 µm; Waters), and the mobile phase was: (A) water containing 0.1% formic acid; or (B) acetonitrile containing 0.1% formic acid. The flow rate was 0.35 mL/min, the analysis time was 12 min, and the column temperature was 40 • C. The gradient elution had the following profile: 0-0.01 min, 99% B; 0.01-1.0 min, 99% B; 1.0-8.0 min, 100% B; 8.0-9.0 min, 100% B; 9.0-9.5 min, 99% B; 9.5-12.0 min, 99% B. The eluents passing through the column were analyzed by QTOF-MS with a negative or positive electrospray ionization (ESI) mode. The QTOF-MS conditions in negative and positive modes were capillary and sampling cone voltages of 2.5 kV/3 kV and 20 V/40 V, respectively, a desolvation flow rate of 900 L/h/800 L/h, a desolvation temperature of 400 • C, and source temperature of 100 • C. QTOF-MS data were analyzed at a scan range of 100-1500 m/z and a scan time of 0.2 s, and leucine-enkephalin was used as a reference compound for the lock mass. MS/MS spectra were obtained under the conditions of a collision energy ramp (10-30 eV) with m/z of 50-1500. Mass spectrometry data processing, including m/z, retention time, and ion intensity, was performed using UNIFI software, version 1.8.2.169 (Waters). Material identification was conducted through online databases connected to UNIFI software.

Ligand and Target Protein Preparation
In this study, only eight of the bioactive compounds identified in OFC and OF extracts by LC-MS analysis were selected for docking studies. Chemical structures of selected bioactive compounds were retrieved through NCBI's PubChem compound database. Twodimensional structures were obtained from NCBI's PubChem compound database, and ligands were prepared using UCSF Chimera. Before proceeding with docking assays, Toll-like receptor 4 (TLR4, PDB ID: 2Z62) and mitogen-activated protein kinase (MAPK, PDB ID: 5MTY) proteins were purified and energy optimized. The study was converted into a protein model using UCSF Chimera software (version 1.16). Finally, PyMol software (version 2.1.0) was used to analyze the protein structures, hydrogen-bonding interactions, and non-bonding interactions of ligands and active site residues and to prepare highresolution images.

Molecular Docking
UCSF Chimera and AutoDock-Vina software were used to dock the anti-inflammatory target proteins TLR4 and MAPK with selected functional compounds and calculate binding energies. Hydrogen atoms, force fields (CHAEMm), and atomic charges were added. The binding energy and binding contacts of each ligand were obtained, and the docked complex was analyzed using PyMol.

2.5.
In-Vitro Anti-Inflammatory Activity 2.5.1. Cell Culture and Viability Assay RAW 264.7 murine macrophage cells (Korean Cell Line Bank, Seoul, Republic of Korea) were cultured using DMEM containing 10% FBS and 1% pen-strep solution and maintained in a humidified incubator at 37 • C with 5% carbon dioxide (CO 2 ). The cells were seeded into a 96-well plate (4 × 10 4 cells/well) and incubated for 24 h. Samples were dissolved in DMSO and incubated with the cells at various doses for an additional 24 h. Subsequently, 0.1 µL/mL of EZ-Cytox solution was added to each well and incubated at 37 • C for 3 h. Then, the absorbance was confirmed at 450 nm on a microplate reader (Infinite M200 Pro; Tecan, Krems, Austria).

Nitric Oxide (NO) Assay
The cells were seeded into a 96-well plate (4 × 10 4 cells/well) and incubated for 24 h. Samples were dissolved in DMSO and pre-incubated with the cells at various doses for 1 h and then stimulated with LPS (1 µg/mL) for an additional 24 h. The cell culture supernatants were mixed with Griess reagent at a ratio of 1:1 and incubated at room temperature for 15 min. Absorbance at 540 nm was confirmed using a microplate reader.

Enzyme-Linked Immunosorbent Assay
The cell culture supernatants were collected, and the levels of cytokines (TNF-α, IL-6, and IL-1β) and PGE 2 were quantified according to the manufacturer's instructions using an ELISA kit. The absorbance of the reacted samples was measured at 450 nm using a microplate reader.

Experimental Data Analysis
All of the data in this study were analyzed using SPSS software (version 20.0; IBM Corp., Armonk, NY, USA). Comparisons were performed using ANOVA with Duncan's multiple range test and independence tests. A p-value < 0.05 was considered to indicate statistical significance.

Plant Materials Used in This Study
In this study, we isolated OFC (fresh callus before extraction and lyophilization) and OF (5 years old and dried cladode powder) ( Figure 1).

2.5.
In-Vitro Anti-Inflammatory Activity 2.5.1. Cell Culture and Viability Assay RAW 264.7 murine macrophage cells (Korean Cell Line Bank, Seoul, S were cultured using DMEM containing 10% FBS and 1% pen-strep solution tained in a humidified incubator at 37 °C with 5% carbon dioxide (CO2). Th seeded into a 96-well plate (4 × 10 4 cells/well) and incubated for 24 h. Sampl solved in DMSO and incubated with the cells at various doses for an addition sequently, 0.1 µL/mL of EZ-Cytox solution was added to each well and incuba for 3 h. Then, the absorbance was confirmed at 450 nm on a microplate rea M200 Pro; Tecan, Krems, Austria).

Nitric Oxide (NO) Assay
The cells were seeded into a 96-well plate (4 × 10 4 cells/well) and incuba Samples were dissolved in DMSO and pre-incubated with the cells at variou h and then stimulated with LPS (1 µg/mL) for an additional 24 h. The cell cu natants were mixed with Griess reagent at a ratio of 1:1 and incubated at roo ture for 15 min. Absorbance at 540 nm was confirmed using a microplate read

Enzyme-Linked Immunosorbent Assay
The cell culture supernatants were collected, and the levels of cytokines 6, and IL-1β) and PGE2 were quantified according to the manufacturer's instru an ELISA kit. The absorbance of the reacted samples was measured at 450 microplate reader.

Experimental Data Analysis
All of the data in this study were analyzed using SPSS software (versio Corp., Armonk, NY, USA). Comparisons were performed using ANOVA wi multiple range test and independence tests. A p-value < 0.05 was considered statistical significance.

Plant Materials Used in This Study
In this study, we isolated OFC (fresh callus before extraction and lyophil OF (5 years old and dried cladode powder) ( Figure 1).

Bioactive Compounds of OFC and of Extract
Cactus species are of scientific and commercial value and have a wide r logically active constituents. There have been reports of potential antioxidant recently studied plant species. However, there is little information about sap vonoid compounds. For this study, it was necessary to determine the biolog compounds. Chromatograms of bioactive components identified in OFC an UPLC-QTOF-MS are shown in Figure S1

Bioactive Compounds of OFC and of Extract
Cactus species are of scientific and commercial value and have a wide range of biologically active constituents. There have been reports of potential antioxidant activities in recently studied plant species. However, there is little information about saponin or flavonoid compounds. For this study, it was necessary to determine the biologically active compounds. Chromatograms of bioactive components identified in OFC and OF using UPLC-QTOF-MS are shown in Figure S1.

Identification of OFC Metabolite Components
In OFC extract, 10 species (four triterpenoids, two fatty acids, one ester, and three steroids) were identified in the negative ion (NEG, Figure S1A), and nine species (four triterpenoids, one antifungal, two fatty acids, and two fatty amides) were identified in the positive ion (POS, Figure S1B). The metabolites identified in OFC consist of a chromatogram ( Figure S1A,B), a component index (Table S1), and component fragments ( . This compound is found in the fruiting bodies of Ganoderma colossum, mainly collected from the stems of Delonix regia (Fabaceae) in Thua Thien-Hue province, Vietnam, and it has been reported as an inhibitor of HIV-1 protease and SARS-CoV-2 major protease [9]. . Triterpenoids such as pachymic acid are major components of Poria cocos, and anti-inflammatory, anti-cancer, and immunomodulatory actions have been reported [12,13]. Peak 10 ([M-H] − ) was identified as eburicoic acid (m/z 469.3676, C 31 H 50 O 3 ). These triterpenoid substances have the potential to protect the liver against CCl4-induced liver damage [14,15].  [16]. It is known that the senescencesuppressing effect of LPE (18:2) inhibits the action of phospholipase D, which mediates the degradation of membrane phospholipids in the early stage of plant senescence [17].

Network and Interpretation of Phytochemicals in OFC Extract
Saponins are secondary metabolites found in various plant species (such as beans, grains, seeds, bark, tea leaves, and root crops), and they protect against attack by potential pathogens [20,21]. Due to their toxicity to various organisms, saponins can be utilized for their antioxidant, antibiotic, fungicidal, anti-inflammatory, and pharmacological properties [22]. The biosynthetic pathway of saponins (a graphical abstract is provided), together with the major carbon source acetyl-CoA, produces a variety of derivatives essential for a number of cellular functions. Various secondary metabolites derived from the mevalonate (MVA) pathway affect sterol synthesis, growth, defense responses, and development [23]. On the other hand, MVA cannot exclude the interaction of the extrinsic pathway (2-C-methyl-D-erythritol 4-phosphate, MEP) with artificial culture. According to the study of Arabidopsis thaliana in medium, the rise in sucrose increased the substrate for MEP action by activity-induced SnRK1 (sucrose non-fermenting 1-related protein kinase 1) phosphorylation and the light-induced metabolism by light-induced IPP. It has been reported to increase MEP-derived terpenoids by increasing dimethylallyl pyprophosphate (DMAPP) production [24]. It can be said that biosynthetic patterns can vary depending on environmental conditions. In organisms, MVA is phosphorylated twice and then decarboxylated to form iso-pentenyl pyrophosphate (IPP), the basic backbone of isoprenoid biosynthesis [25]. Both IPP and DMAPP are used as synthetic intermediates. Then, squalene synthase, a key enzyme in the biosynthesis of the carbon ring backbone in the middle of triterpene saponins, catalyzes the internal cyclization of two molecules of farnesyl diphosphate to yield one molecule of squalene [26]. As triterpenoid glycosides, they have a 30C oxidosqualene precursor and are linked to glycosyl residues to form saponins [27,28]. Subsequent conversion into squalene forms various triterpenoid and fungal sterol compounds from 2,3-oxidosqualene. The physiological activity of the plant saponin is influenced by its chemical structure. Saponin glycosyl groups hydrolyzed by physical, chemical, and microbial transformations are converted into rare saponins (with short sugar chains) that are highly absorbed by the body. For example, it has been reported that saponin, an insoluble compound of ginseng, is converted by various biomaterials, and its physiological activity is modified [29]. However, it is clear that some saponins have side effects (allergies or gastrointestinal problems) when administered in high doses [30]. For cactus callus, it is believed that further research on component changes and mechanisms through additional processing is required.

Identification of OF Metabolite Components
In OF extract, 11 species (two phenolic compounds, nine flavonols, one flavanone, and one flavone) were identified in NEG (Figure S1C), and 12 species (one triterpenoid, nine flavonols, and two flavones) were identified in POS ( Figure S1D). The metabolites identified in OF are composed of a chromatogram ( Figure S1C,D), a component index (Table S2), and component fragments (Table S4).   ). This compound has been isolated and reported from Astragalus membranaceus, and its efficacy includes in colds, upper respiratory tract infections, fibromyalgia, and diabetes [31]. . This aglycone has shown strong pharmacological effects, such as anti-oxidant, anti-fungal, anti-asthmatic, anti-inflammatory, and anti-convulsant effects, in several in-vitro studies, and it has been reported as a substance with anti-osteoporotic activity, proven to be similar to estrogen [38].

Network and Interpretation of Phytochemical in OF Extract
The flavonoid compounds of each peak (including NEG and POS) were identified as glycones, except for eriodictyol, quercetin, isorhamnetin, kaempferol, and hispidulin, which are aglycones. The biosynthesis of secondary metabolites starts with the condensation of erythrose-4-phosphate and phosphoenolopyruvate to produce shikimic acid, a metabolite of aromatic amino acid biosynthesis [39]. Phenylalanine is converted into trans-cinnamic acid by PAL, and CA4H catalyzes the conversion to p-coumaric acid by the hydroxy group of the phenyl ring of cinnamic acid. The COOH of cinnamic acid forms a thioester bond with CoA, a process catalyzed by 4-coumarate-CoA ligase [40]. Chalcone synthase catalyzes the formation of chalcone by the condensation of one p-coumaric-CoA with three malonyl CoAs. Naringenin, a group of flavanones produced by CHI, is formed and converted into flavonols and flavones by the catalysis of F3H, FLS, and DFR, respectively. The phytochemical compounds of the cactus cladode have attracted attention as useful functional substances that can improve human health. Because of the high fiber content of cactus cladodes, trace compounds, such as phenolic and flavonoid compounds among phytochemicals, have not been studied in depth. On the other hand, in regions with tropical climates, including South America, it has been studied as a useful plant material for preventing metabolic diseases (obesity, diabetes, and cardiovascular disease). Traditional Mexican medicine recommends consuming cladodes due to the health effects of their bioactive compounds. The biological activities of cactus cladodes studied include anti-inflammatory, anti-diabetic, antibacterial, antioxidant, hyperlipidemic, neuroprotective, and immunoprotective activities [41]. The main antioxidant mechanism is hydrogen atom transfer by phenolic OH groups. The antioxidant activity of flavonoid compounds is influenced by specific chemical reaction patterns. The reaction patterns are the hydroxyl bond pattern and the catechol structure (substitution of 3 , 4 OH groups of the B-ring), and they are also influenced by the level of conjugation sites allowed [42]. This flavonoid binding structure increases the degree of activity through resonance stabilization of radicals and induces interactions between compounds in the OF extract. The major flavonoid antioxidant activity has been reported at the levels of quercetin, kaempferol, and myricetin > rutin > luteolin > taxifolin by substitution and binding patterns. However, it is still unclear how to reflect absorption and metabolism with only known patterns [42,43]. To fractionate polymeric antioxidants from frozen and oven-dried cactus cladodes samples, acid hydrolysis treatment of the residue, sequentially extracted with methanol, acetone, and distilled water, increased antioxidant content by about eight times compared to conventional acid hydrolysis [44]. Additionally, the identification and detection of piscidic acid, quercetin, kaempferol, and isorhamnetin components were reported. They were similar to the phenolic and flavonoid compounds of cactus cladodes identified by LC-MS in this study.

Molecular Docking Results and Interpretation
To predict the overall physiological activity of each extract, some specific anti-inflammatory compounds were selected from the metabolites profiled. The selected compounds were subjected to molecular docking assays to confirm their binding interactions with inflammatory response receptors. Toll-like receptor 4 (TLR4) and mitogenactivated protein kinase (MAPK) are important receptors in the LPS-induced inflammatory response. In general, cell signaling pathways in the regulation of inflammation are complex and involve cross-talk, but the interactions between phytochemicals and protein receptors are related to their bioactivities. The interaction results between the target protein and the bioactive compound ligand selected from OFC and OF extracts are as follows (Table S5). Docked saponin compound ligands showed binding energies ranging from −6.2 to −6.6 Kcal/mol for the TLR4 protein and binding energies ranging from −7. These results indicate that the affinity with MAPK protein is greater than the affinity with TLR4 protein. A higher negative value (affinity or binding energy) in a docking study indicates greater affinity between the target protein and the ligand molecule, indicating greater efficiency of the bioactive compound. The affinity between a protein and a ligand for the van der Waals term is known to be about −15.0 Kcal/mol at most in biophysical binding, and about −9.0 Kcal/mol has been interpreted as a normal distribution [45]. The visualized molecular docking 3D interaction diagram is shown in Figures 2 and 3. Complex compounds extracted from plants are considered predictable only when many variables and diverse of compound types are systematically calculated to confirm their applicability to the human body before being used as food through molecular dynamics or docking analysis. To un-derstand the plausible experimental anti-inflammatory activity of the compounds currently studied, prediction of inhibitory dissociation constant (Ki) values was performed. The predicted Ki (pKi) value indicates how potent a compound is as an inhibitor of a biological process, and it is used as a universal number to symbolize the concentration required to produce half-maximal inhibition [46,47].

Anti-Inflammatory Activity of OFC and of Extract
In-vitro anti-inflammatory activities of OFC and OF extracts were compared. RAW 264.7 cells were treated with two samples (OFC and OF in 70% ethanol extracts) at various concentrations for 24 h, and their cytotoxicity was evaluated using the EZ-Cytox reagent. No cytotoxicity was observed in either sample up to a concentration of 200 µg/mL ( Figure 4A). The concentrations of the two samples used in the activity experiment were determined to be 25, 50, 100, and 200 µg/mL. LPS treatment showed a significant decrease in NO production in a dose-dependent manner (p < 0.001, Figure 4B). In addition, in the comparison between the two samples, the activity of OF was significantly lower than that of OFC (p < 0.001). The production amount of PGE 2 was significantly increased compared to the control group (p < 0.001). In the comparison of the samples, OFC was 9.35 ng/mL, OF was 8.44 ng/mL at a concentration of 200 µg/mL, and the production of OFC was significantly decreased (p < 0.01). In addition, LPS-treated cells produced pro-inflammatory cytokines, such as TNF-α, IL-6, and IL-1β, compared to the control group. On the other hand, NO, PGE 2 , and pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β) increased by LPS were significantly decreased in a dose-dependent manner in cells pretreated with each sample extract (p < 0.05, Figure 4C-F). Pro-inflammatory cytokine factors at a concentration of 200 µg/mL included LPS-stimulated TNF-α (OFC 72.33 ng/mL, OF 66.78 ng/mL) and IL-1β (OFC 49.10 pg/mL, OF 34.45 pg/mL), both of which significantly decreased OF (p < 0.01, p < 0.001). In particular, at a concentration of 50 µg/mL or more, cacti reduced the production of IL-1β by LPS stimulation more effectively than OFC (western blotting results for expression changes are not shown). In addition, the anti-inflammatory effects of cacti have been reported in the past [48]. Antioxidants 2023, 12, x FOR PEER REVIEW 12 of 15

Conclusions
This study showed that OFC and OF extracts grown in Korea can be considered secondary metabolite sources involved in the inhibition of LPS-induced inflammatory responses. A qualitative analysis allowed for the identification of saponin and phenolic compounds related to triterpenoids and flavonol glycosides. Exploration of the biological activity of the studied extracts showed that OFC and OF possess antioxidant and anti-inflammatory effects. From the results obtained, it seems feasible to use OFC and OF as sources of natural compounds that could be included in foods, cosmetics, and pharmaceuticals. These findings could stimulate research to elucidate the mechanism of action of plant saponins and flavonoids with similar structures, including those with antioxidant and anti-inflammatory properties. The data are shown as the mean ± SD (n = 3). ### p < 0.001 compared with the control (none) group; * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with LPS-stimulated RAW 264.7 cells and treatment groups; $ p < 0.05, $$ p < 0.01, and $$$ p < 0.001 compared by sample group.

Conclusions
This study showed that OFC and OF extracts grown in Korea can be considered secondary metabolite sources involved in the inhibition of LPS-induced inflammatory responses. A qualitative analysis allowed for the identification of saponin and phenolic compounds related to triterpenoids and flavonol glycosides. Exploration of the biological activity of the studied extracts showed that OFC and OF possess antioxidant and antiinflammatory effects. From the results obtained, it seems feasible to use OFC and OF as sources of natural compounds that could be included in foods, cosmetics, and pharmaceuticals. These findings could stimulate research to elucidate the mechanism of action of plant saponins and flavonoids with similar structures, including those with antioxidant and anti-inflammatory properties.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/antiox12071329/s1, Figure S1: UPLC-QTOF-MS metabolite component profile; Table S1: Identification of metabolite components in OFC extract; Table S2: Identification of metabolite components in OF extract; Table S3: Identification of metabolite component fragments in OFC extract; Table S4: Identification of metabolite component fragments in OF extract; Table S5: Energy of complexes formed between selected compounds and target protein.