Article Anti-Inflammatory Effect of argyi on Ethanol-Induced Gastric Ulcer: Analytical, In Vitro and In Vivo Studies for the Identification of Action Mechanism and Active Compounds

Myoung-Sook Shin 1 , Jaemin Lee 1, Jin Woo Lee 2, Se Hoon Park 2, Il Kyun Lee 2, Jung A. Choi 2, Jung Suk Lee 2 and Ki Sung Kang 1,*

1 Korean Medicine, Gachon University, Seongnam-si, Gyeonggi-do 13120, Korea; [email protected] (M.-S.S.); [email protected] (J.L.) 2 Research & Development Center, Richwood Pharmaceuticals, 1, Gwanak-gu, Seoul 08826, Korea; [email protected] (J.W.L.); [email protected] (S.H.P.); [email protected] (I.K.L.); [email protected] (J.A.C.); [email protected] (J.S.L.) * Correspondence: [email protected]; Tel.: +82-31-750-5402

Abstract: Artemisia argyi is widely used as traditional medicine in East Asia. However, its effects against inflammation and gastric ulcers have not been reported yet. We analyzed anti-inflammatory activity and its molecular mechanisms of A. argyi using RAW264.7 cells line, then evaluated the curative efficacy in rats with acute gastric ulcers. Nitric oxide and IL-6 production was measured using Griess reagent and an ELISA kit. Inducible nitric oxide synthase (iNOS), interleukin (IL)-6, and mucin (MUC)1, MUC5AC, and MUC6 mRNA were determined by SYBR Green or Taqman

 qRT-PCR methods. The phosphorylation of ERK, JNK, p38, and c-Jun protein were detected by  western blotting. RW0117 inhibited LPS-induced NO and IL-6 production. The mRNA levels of

Citation: Shin, M.-S.; Lee, J.; Lee, J.W.; iNOS and IL-6 were strongly suppressed. The phosphorylation of ERK, JNK, and c-Jun decreased Park, S.H.; Lee, I.K.; Choi, J.A.; Lee, J.S.; by treatment with RW0117. Oral administration of RW0117 recovered the amount of mucin mRNA Kang, K.S. Anti-Inflammatory Effect and protein level that was decreased due to gastric ulcers by HCl-EtOH. A. argyi exhibited strong of Artemisia argyi on Ethanol-Induced anti-inflammatory effects and contributed to the modulation of HCl-EtOH-induced gastric ulcer Gastric Ulcer: Analytical, In Vitro and in rats. In Vivo Studies for the Identification of Action Mechanism and Active Keywords: Artemisia argyi; anti-inflammation; antigastric ulcer Compounds. Plants 2021, 10, 332. https://doi.org/10.3390/plants10020332

Academic Editor: Adam Stebel 1. Introduction Received: 23 December 2020 Inflammation is an immune response of a living organism against external stimuli Accepted: 4 February 2021 Published: 9 February 2021 or tissue damage, and it is a complex process involving various immune cells. However, excessive inflammatory reactions can cause chronic inflammatory diseases; corresponding

Publisher’s Note: MDPI stays neutral responses to inflammatory reactions in the human body are closely related to the preven- with regard to jurisdictional claims in tion or occurrence of diseases [1,2]. Phagocytosis or secretion of inflammatory mediators published maps and institutional affil- is a well-known inflammatory reaction by immune cells such as macrophages exposed iations. to viruses or pathogens [3]. Macrophages play an important role in innate and acquired immune responses. Macrophages are activated by lipopolysaccharide (LPS) that produces inflammatory mediators such as nitric oxide (NO), prostaglandin E2, tumor necrosis factor- α (TNF-α), interleukin-6 (IL-6), and IL-1β [4,5]. LPS, also known as endotoxin, exists in the outer membrane of gram-negative bacteria and activates the transcription factor Copyright: © 2021 by the authors. κ κ Licensee MDPI, Basel, Switzerland. nuclear factor- B (NF- B), thereby activating inducible nitric oxide synthase (iNOS) and This article is an open access article cyclooxygenase-2 (COX-2) and ultimately producing the respective inflammatory media- distributed under the terms and tors [6]. Acute gastritis is a disease characterized by inflammation of the gastric mucosa conditions of the Creative Commons due to stress, smoking, alcohol, irregular meals, or prolonged use of nonsteroidal anti- Attribution (CC BY) license (https:// inflammatory drugs (NSAIDS) [7–9]. When this disease occurs, it causes gastrointestinal creativecommons.org/licenses/by/ discomfort and loss of the gastric mucosa due to vomiting. The gastric mucosa has compo- 4.0/). nents such as mucus, bicarbonates, and prostaglandins that protect the stomach against

Plants 2021, 10, 332. https://doi.org/10.3390/plants10020332 https://www.mdpi.com/journal/plants Plants 2021, 10, 332 2 of 13

gastric lesions [10]. In clinical practice, proton pump inhibitors and H2 receptor antagonists are used to treat ulcers, but these drugs cause side effects and recurrence of gastritis has been observed after treatment [11]. Alcohol is one of the most common pathogenic factors of gastric injury [7]. Excessive alcohol consumption can weaken the protective function of the gastric mucosa. Therefore, ethanol-induced gastric ulcer animal models are often used to screen for compounds that possess antiulcer activity [12–14]. Previous studies already reported that extracts and plant-derived compounds such as Allophylus serratus, Aloe vera and kaempferol (3,5,7,40-tetrahydroxy flavone) showed anti-inflammatory and antigastric ulcer effects in in vitro and in vivo [15–17]. Artemisia argyi (A. argyi) has been traditionally used in , Japan, and Korea to treat conditions such as inflammation, abdominal pain, hemorrhage, hepatitis, and liver cirrhosis [18]. Recent studies have shown that A. argyi extracts reduce symptoms of allergic asthma and dermatitis in vivo and possess immunosuppressive activity [19–21]. The flavonoids eupatilin, jaceosidin, hispidulin, and caffeoylquinic acid present in A. argyi are responsible for its pharmacological effects [22–24]. In this study, we prepared an extract of A. argyi (RW0117) and analyzed its anti- inflammatory activity and identified its intracellular signaling pathways. We also evaluated its antigastric ulcer activity in rats.

2. Materials and Methods 2.1. Chemicals and Reagents Antibodies against p65 (C-20), p38 (C-20), ERK1 (C-16), JNK (FL), and β-actin (I-19) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against phospho-p65 (Ser-536), phospho-ERK1/2 (Thr202/Tyr204), phospho-JNK (Thr-183/Tyr- 185), phospho-p38 (Thr-180/Tyr-182), phospho-c-Jun (Ser-73), c-Jun (60A8), and inducible NO synthase (iNOS, D6B6S) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Ultrapure LPS from 0111:B4 was obtained from Invitrogen (San Diego, CA, USA). Dulbecco’s modified eagle’s medium (DMEM) was purchased from Hyclone (GE Healthcare Life Sciences, Chicago, IL, USA). Fetal bovine serum (FBS) was purchased from ATCC (Manassas, VA, USA). Eupatilin (CAS No.: 22368-21-4), jaceosidin (CAS No.:18085-97-7) and hispidulin (Cas No.:1447-88-7) were purchased from Sigma (St. Louis, MO, USA). All other chemicals and reagents were purchased from Sigma (St. Louis, MO, USA). Beeswax alcohol (Abexol®) was used as the positive control material and was purchased from Rainbow & Nature Pty Ltd. (Seoul, Korea).

2.2. Plant Material and Extraction Artemisia argyi H.Lev. & Vaniot (A. argyi) was collected in Jiangsu Province in China (2018, June), and was purchased as dried material from NINGBO SIMINGSHAN BIO- TECHNOLOGY (Ningbo, Zhejiang, China) (2018, Sept.) Raw material was identified by comparing database of the National Center for Biotechnology Information (NCBI, USA) after DNA sequence analysis (Table S1). A total of 16 L of 66.5% ethanol was added to 1 kg of A. argyi to perform cold extraction for 24 h. The extract was passed through a filter and concentrated under reduced pressure at <60 ◦C until a volume 400 mL of extract was obtained. This concentrated solution was dried under reduced pressure at <70 ◦C, and then pulverized to obtain 98.2 g of the dried product, RW0117 (yield: 9.82%).

2.3. HPLC-UV/DAD Conditions Quantitative analysis of RW0117 was performed using an Agilent 1260-DAD system (Santa Clara, CA, USA) and Inno column C18 (250 mm × 4.6 mm, 5 µm) Santa Clara, CA, USA). The mobile phase consisted of water containing 0.05% trifluoroacetic acid (A) and acetonitrile containing 0.05% trifluoroacetic acid (B). The gradient elution was as follows: 0–30 min of 30% (B), 30–31 min of 60–100% (B), 31–36 min of 100% (B), 36–37 min of 100–30% (B), 37–42 min of 30% (B) The post-running time was 10 min after restoration of the initial condition. The mobile phase flow rate was 0.7 mL/min and the injection Plants 2021, 10, 332 3 of 13

volume was 10 µL. A PDA eλ detector was set to an absorbance of 340 nm for eupatilin, jaceosidin and, hispidulin. The peaks for eupatilin, jaceosidin and, hispidulin in RW0117 were compared with their respective standard compounds.

2.4. Cell Culture RAW264.7 murine macrophage cells were obtained from the Korean Cell Line Bank (Seoul, Republic of Korea) and cultured at 37 ◦C in DMEM supplemented with 10% heat- inactivated fetal bovine serum (FBS) and 1% penicillin/streptomycin in a humidified atmosphere with 5% CO2 and 95% air. The cells were subcultured every 2 days to maintain monolayer cells.

2.5. Measurement of RW0117 Cytotoxicity on RAW264.7 Cells The cytotoxic effect of RW0117 in RWA264.7 cells was examined using an EZ-Cytox enhanced cell viability assay kit. Cells were seeded onto 96-well plates and treated with ◦ various concentrations of RW0117 for 24 h at 37 C in a humidified atmosphere of 5% CO2 and 95% air.

2.6. Determination of Nitric Oxide, IL-6, and TNF-α Production RAW264.7 cells were treated with RW0117 (200 µg/mL, 100 µg/mL, 50 µg/mL, 25 µg/mL, 12.5 µg/mL, 6.3 µg/mL, 3.1 µg/mL, 1.6 µg/mL, 0.8 µg/mL) for 2 h followed by treatment with LPS for 20 h in a CO2 incubator. The positive control consisted of macrophage cells treated with LPS alone for NO and IL-6 production. The supernatants were collected after centrifugation (900 rpm for 3 min) and frozen at −80 ◦C for further analysis. NO concentration in the supernatants was measured using a Griess reagent system (Promega, WI, USA), whereas IL-6 was measured using a kit for sandwich ELISA from eBioscience (San Diego, CA, USA), according to the manufacturer’s instructions.

2.7. Immunoblotting RAW 264.7 cells were treated RW0117 (100 µg/mL, 50 µg/mL) for 2 h and then treated with LPS for 30 min (for phosphorylation of c-Jun, EKR, JNK, p38, p65) or 20 h (for protein expression of iNOS and COX-2). Following treatment, the cells were washed with cold PBS and lysed with cold radioimmunoprecipitation assay (RIPA) buffer containing a protease inhibitor cocktail (Roche Diagnostics Corp., Indianapolis, IN, USA), 1 mM dithiothre- itol (Wako, Tokyo, Japan), 1 mM phenylmethylsulfonyl fluoride (Sigma), 1 mM sodium orthovanadate (Sigma), and 10 mM β-glycerophosphate (Sigma). Cell lysis, collection of su- pernatants, protein quantification, protein electrophoresis, protein transfer, and membrane development were all performed as described in our previous report [25,26]. The protein bands were visualized using an enhanced chemiluminescence system (GE Healthcare Life Sciences).

2.8. Real-Time Reverse Transcription Polymerase Chain Reaction RAW 264.7 cells were treated with RW0117 (100 µM, 50 µM) for 2 h, and then treated with LPS for 4 h. Following treatment, the cells were washed with PBS and lysed using the RNeasy mini kit (Qiagen, Valencia, CA, USA), after which, a procedure for total RNA purification was performed according to the manufacturer’s protocol. RNA was converted into cDNA using the RevertAid First Strand cDNA Synthesis kit (Thermo Scientific, Madison, WI, USA). To amplify the cDNA encoding for mouse IL-6, specific primers such as TNF-α and β-actin genes were used (Table1). PCR amplification was performed using Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA). Relative expression levels were determined using real-time reverse transcription PCR (qRT-PCR) by the Quant3 real-time PCR System (Applied Biosystems). Data were normalized to the amount of β-tubulin. Plants 2021, 10, 332 4 of 13

Table 1. The primer sets used for real-time PCR analysis.

Gene Forward Primer (50-30) Reverse Primer (50-30) Mouse IL-6 GAGGATACCACTCCCAACAGACC AAGTGCATCATCGTTGTTCATACA Mouse iNOS ACATCGACCCGTCCACAGTAT CAGAGGGGTAGGCTTGTCTC Mouse β-tubulin CTCCCAGGTTAAAGTCCTTC GCAACATAAATACAGAGGTG Rat MUC1 Rn01462585_m1 (Muc1) Rat MUC5AC Rn01451252_m1 (Muc5ac) Rat MUC6 Rn01759814_m1 (Muc6) Rat GAPDH Rn01775763_g1 (gapdh)

2.9. Immunofluorescence Microscopy To investigate the localization of p65 in RAW264.7 cells treated with LPS or RW0117, the cells were seeded onto gelatin-coated coverslips (Paul Marienfeld GmbH & Co. KG, Lauda-Königshofen, Germany) in a 24-well plate, and grown to 70% confluence. Cells in the experimental group were treated with RW0117 (25 µg/mL and 50 µg/mL) for 2 h and then treated with LPS for 60 min. Subsequently, the cells were washed with PBS and fixed with 4 % formalin (pH 7.2) for 15 min. Additionally, cells were washed three times with PBS and incubated with blocking solution (5% BSA containing 0.1% Trion X-100) for 1 h. fter blocking, the cells were incubated with p65 antibody for 12 h at 4 ◦C, washed three times with PBS, and later incubated with Alexa Fluor 488-conjugated goat antirabbit antibody for 1 h in the dark. After incubation, the cells were again washed three times with PBS. The coverslips were removed from the 24-well plate and mounted onto glass slides containing a drop of mounting medium (DAPI, Vectashield, Burlingame, CA, USA). The fluorescent-labeled cells were analyzed using a confocal laser scanning microscope (Carl Zeiss Microscopy, Oberkochen, Germanay).

2.10. Animals Male Sprague-Dawley rats (7 weeks old) (supplied by OrientBio, Seongnam, Korea) were used for all the experiments. The animals were allowed an acclimation period for 7 days under a controlled temperature of 22 ◦C ± 2 ◦C with a 12 h light/dark cycle and free access to food and water All animal experiments were performed in accordance with the instruction of the Ethics Comminttee for Use of Experimental Animals at Gachon University (Ethical approval code: 2019-032).

2.11. Ethanol and HCl-Induced Gastric Ulcer Acute gastric lesions were induced by oral administration of 0.1 mL/20 g of a mixture containing 0.15 M HCl in 98% ethanol, a dose that induces significant gastric ulceration [27]. After 24 h of food deprivation, groups of animals (n = 5/group) were orally administered different doses of RW0117 (25 mg/kg and 50 mg/kg in 1.5% carboxymethylcellulose solution). Subsequently, 1 h after oral administration of the above solutions, the animals were orally administered 0.2 mL ethanol or the ethanol/HCl mixture, and 1 h later, they were euthanized. Their stomachs were removed, and then incised along the greater curvature. The stomachs were gently rinsed with saline solution to remove the gastric contents and blood clots.

2.12. Histological Analysis A small portion of the stomach of each rat was fixed in 4% formalin solution for 24 h. The sections of the stomach tissue were dehydrated with graded concentrations of ethanol, passed through xylene, and embedded in paraffin. The paraffin sections were stained with periodic acid-Schiff stain [17].

2.13. Statistical Analysis Results of the three independent experiments were expressed as mean ± standard deviation (SD). All statistical analyses were performed using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. ** p < 0.01 or * p < 0.05 indicated significance. Plants 2021, 10, x FORPlants PEER 2021 REVIEW, 10, x FOR PEER REVIEW 5 of 13 5 of 13

2.13. Statistical Analysis2.13. Statistical Analysis Results of the threeResults independent of the three experiments independent were experiments expressed wereas mean expressed ± standard as mean ± standard deviation (SD). Alldeviation statistical (SD). analyses All statistical were performed analyses wereusing performed one-way analysis using one-way of variance analysis of variance Plants 2021, 10, 332 (ANOVA) followed(ANOVA) by Tukey’s followed post hocby Tukey’s test. ** p post < 0.01 hoc or test. * p < ** 0.05 p < indicated0.01 or * p significance.< 0.05 indicated5 of 13 significance.

3. Results and Discussion3. Results and Discussion 3.1.3. ResultsHPLC Profile and3.1. Discussionof RW0117 HPLC Profile of RW0117 3.1.Through HPLC Profile HPLC of RW0117analysisThrough we HPLC showed analysis that weRW0117 showed contained that RW0117 phytochemicals contained phytochemicalssuch as such as eupatilin,Through jaceosidin, HPLCeupatilin, and analysis hispidulin jaceosidin, we showed (Figure and hispidulin that 1). RW0117From (FigureHPLC contained chromatogram 1). From phytochemicals HPLC we chromatogram calculated such as we calculated thateupatilin, RW0117 jaceosidin, containedthat RW0117 and 0.573%, hispidulin contained 0.223%, (Figure 0.573%,and1). 0.070% From 0.223%, HPLC of eupatilin,and chromatogram 0.070% jaceosidin of eupatilin, we calculatedand his-jaceosidin and his- pidulin,that RW0117 respectively containedpidulin, (Table 0.573%,respectively 2). 0.223%,Previous (Table and studies 0.070%2). Previous reported of eupatilin, studies that jaceosidinthese reported three and that compound hispidulin, these three compound showedrespectively anti-inflammatory (Tableshowed2). Previous anti-inflammatory activity studies in vitro reported activityand in that vivoin these vitro [22–24]. threeand inTherefore, compound vivo [22–24]. we showed also Therefore, con- anti- we also con- firmedinflammatory eupatilin, activityfirmed jaceosidin, ineupatilin, vitro andand hispidulinjaceosidin,in vivo [22 showedand–24 ].hispidulin Therefore, the inhibitory showed we also effects the confirmed inhibitory of nitric eupatilin, oxideeffects of nitric oxide productionjaceosidin, by and LPS hispidulinproduction in RAW264.7 showed by LPS cells. thein RAW264.7 inhibitoryAs shown effects cells.in figure As of nitric shown2, treatement oxide in figure production of 2,eupatilin treatement by LPS (200 in of eupatilin (200 μRAW264.7M), jaceosidin cells. (200μ AsM), shownμ jaceosidinM, 100 in μ FigureM, (200 50 2μμ,M, treatement and100 μ25M, μ ofM)50 eupatilin μandM, andhispidulin (20025 μM)µM), (100 and jaceosidin μ hispidulinM, 50 μ (200M, (100 andµM, μM, 50 μM, and 25100 μM)µM, suppression 50 µM,25 and μ M)of 25 LPS-induced suppressionµM) and hispidulin ofnitric LPS-induced oxide (100 productionµM, nitric 50 µ oxideM, in and concentration production 25 µM) suppression in dependent concentration of dependent manner,LPS-induced respectively nitricmanner, oxide(Figure respectively production 2). In addition, (Figure in concentration these 2). In concentrations addition, dependent these did concentrations manner, not show respectively cell did cyto- not show cell cyto- toxicity(Figure in2 ).RAW264.7 In addition,toxicity cells in these (dataRAW264.7 concentrations not shown). cells (data Taken did not nottogether, shown). show it cellTaken was cytotoxicity predicted together, itthat in was RAW264.7 RW0117 predicted that RW0117 couldcells (dataexhibit not anti-inflammatory shown).could exhibit Taken anti-inflammatory together, activity itbeca wasuse predicted activityit contained beca thatuse RW0117eupatilin, it contained could jaceosidin, exhibit eupatilin, and anti- jaceosidin, and hispidulin.inflammatory activityhispidulin. because it contained eupatilin, jaceosidin, and hispidulin.

Figure 1. HPLC profilesFigure of1. RW0117.HPLCFigure profiles 1. TheHPLC peak of RW0117. profiles of eupatilin, ofThe RW0117. peakjaceos ofidin, Theeupatilin, hispidulin peak ofjaceos eupatilin, atidin, 340 nmhispidulin jaceosidin, in RW0117 at hispidulin340 were nm comparedin RW0117 at 340 nm were in compared with those of their respective standard compounds. with those of their respectiveRW0117 standard were compounds. compared with those of their respective standard compounds.

Figure 2. The inhibitoryFigure 2.effects TheFigure inhibitoryof 2.eupatilin,The inhibitory effects jaceosidin of effectseupatilin, and of hispidulin eupatilin, jaceosidinjaceosidin on and nitric hispidulin andoxide hispidulin production on nitric on nitricoxidein LPS-stimulated oxideproduction production in LPS-stimulated in RAW264.7 cells; (−RAW264.7): vechicle LPS-stimulatedgroupcells; ( −(0.1%): vechicle DMSO-treat RAW264.7 group (0.1%ed cells; group), DMSO-treat (−): vechicle(+): positiveed group group), control (0.1% (+): group DMSO-treated positive (500 control ng/mL group), group of LPS (+): (500 treatment positive ng/mL control of LPS treatment for 20 h), Dexa: dexamethasonefor 20 h), Dexa:group for dexamethasone 2(500 h and ng/mL then incubated of for LPS 2 h treatmentand with then LP incubatedS forfor 20 h. h), Cellswith Dexa: LPwere dexamethasoneS for treated 20 h. Cellswith eupatilinwere for 2 treated h and or jaceosidin with then eupatilin incubated or jaceosidin or hispidulin for 2or h hispidulinand thenwith incubated for LPS 2 h forand with 20 then h.LPS incubated Cells for 20 were h. withRe treatedsults LPS are with for expressed 20 eupatilin h. Results as or mean are jaceosidin expressed ± SD of or triplicate hispidulinas mean experiments.± SD for of 2 htriplicate and then experiments. Statistical significance was determined using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. * Statistical significance was determinedincubated withusing LPS one-way for 20 analysis h. Results of are variance expressed (ANOVA) as mean followed± SD of by triplicate Tukey’s experiments. post hoc test. Statistical * p < 0.001 vs. LPS treated group. p < 0.001 vs. LPS treated group.significance was determined using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. * p < 0.001 vs. LPS treated group. Plants 2021, 10, 332 6 of 13

Plants 2021, 10, x FOR PEER REVIEW 6 of 13

Table 2. Contents of representative compounds in RW0117.

ActiveActive Compound Compound Content Content (%) (%) EupatilinEupatilin 0.573 0.573± 0.002 ± 0.002 JaceosidinJaceosidin 0.223 0.223± 0.006 ± 0.006 HispidulinHispidulin 0.070 0.070± 0.002 ± 0.002

3.2.3.2. The The Effects Effects of of RW0117 RW0117 on Nitric Oxide Production in LPS-Stimul LPS-Stimulatedated RAW 264.7 Cells TreatmentTreatment of of RAW264.7 RAW264.7 cells cells with with LPS LPS (an (an inflammatory inflammatory substance) substance) leads leads to the to pro- the ductionproduction of proinflammatory of proinflammatory medi mediatorsators (nitric (nitric oxide, oxide, IL-6, IL-6, IL-1 IL-1ββ, and, and TNF- TNF-αα).). To To confirm confirm thethe inhibition ofof LPS-inducedLPS-induced inflammationinflammation by by RW0117, RW0117, we we investigated investigated the the production production of ofinflammatory inflammatory mediators. mediators. First, First, the the cytotoxicity cytotoxicity of RW0117of RW0117 was was confirmed confirmed in the in the presence pres- enceof LPS of LPS and and RW0117. RW0117. As As described described in in the the experimental experimental method method section, section, thethe cellscells were pretreated with RW0117 for 2 h and then incubatedincubated with LPS for 20 h.h. The cytotoxicity was confirmed confirmed in the group treated with 200 µμg/mL of of RW0117; RW0117; however, however, no no cytotoxicity cytotoxicity was observed at a concentration of <200<200 µμg/mL (Figure (Figure 33).). Next,Next, thethe inhibitoryinhibitory effecteffect ofof RW0117RW0117 on nitric oxide (NO) production wa wass analyzed. LPS-induc LPS-induceded NO production was significantlysignificantly inhibited at 25, 50, and 100 μµg/mL of of RW0117 RW0117 treatment treatment (Figure (Figure 33).).

FigureFigure 3. TheThe effects effects of of RW0117 RW0117 on on nitric nitric oxide oxide prod productionuction in in LPS-stimulated LPS-stimulated RAW264.7 RAW264.7 cells cells;; (− (−): ): vechicle group (0.1% DMSO-treatedDMSO-treated group),group), (+): (+): positivepositive controlcontrol group group (500 (500 ng/mL ng/mL ofof LPSLPS treatment treat- mentfor 20 for h). 20 Cells h). Cells were were treated treated with RW0117with RW0117 for 2 hfor and 2 h then and incubatedthen incubated with LPSwith for LPS 20 for h. Results20 h. Re- are sults are expressed as mean ± SD of triplicate experiments. Statistical significance was determined expressed as mean ± SD of triplicate experiments. Statistical significance was determined using using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. * p < 0.001 vs. one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. * p < 0.001 vs. LPS treated LPS treated group. group.

Plants 2021, 10, 332 7 of 13

Plants 2021, 10, x FOR PEER REVIEW 7 of 13

3.3.3.3. Analysis Analysis of of the the Effect Effect of of RW0117 RW0117 on on Cytokine Cytokine Production Production and and mRNA mRNA Expression Expression in in RAW264.7RAW264.7 Cells Cells TheThe inhibitory inhibitory effects effects RW0117 RW0117 against against NO NO and and IL-6 IL-6 production production were were analyzed analyzed using using NSAIDNSAID (dexamethasone, (dexamethasone, Dexa.) Dexa.) and and beeswax beeswax alcohols alcohols (Beeswax) (Beeswax) (Abexol (Abexol®, Rainbow®, Rainbow & Nature& Nature Pty PtyLtd., Ltd., Seoul, Seoul, Korea). Korea). as positive as positive controls. controls. Beeswax Beeswax (a mixture (a mixture of six high of six molec- high ularmolecular weight weight aliphatic aliphatic alcohols alcohols purified purified from frombeeswax), beeswax), has been hasbeen shown shown gastroprotective gastroprotec- effectstive effects in experimental in experimental and clinical and clinical studies studies [28]. As [28 ].shown As shown in Figure in Figure 4, NO4, and NO IL-6 and pro- IL-6 ductionproduction induced induced by LPS by LPSwas wassignificantly significantly inhibi inhibitedted in the in group the group treated treated with with Dexa. Dexa. as the as positivethe positive control. control. Additionally, Additionally, in the in the group group treated treated with with 100 100 μµg/mLg/mL of of beeswax beeswax alcohol, alcohol, thethe inhibitory inhibitory activity activity due due on on NO NO and and IL-6 IL-6 production production displayed displayed similar similar with with the the Dexa. Dexa. group.group. In In the the group group treated treated with with 50 50 μµg/mLg/mL of of beeswax beeswax alcohol, alcohol, no no inhibitory inhibitory activity activity was was observed.observed. Conversely, Conversely, in in the the case case of of RW0117, RW0117, there there was was a a significant significant inhibitory inhibitory effect effect on on LPS-inducedLPS-induced NO NO production production at at all all concentrations concentrations (6.3–100 μµg/mL), while while IL-6 IL-6 production production waswas significantly significantly inhibited inhibited at at 100 100 μµg/mLg/mL of of RW0117 RW0117 (Figure (Figure 4).4). Next, Next, the the mRNA mRNA expres- expres- sionsion of of iNOS iNOS and and IL-6, IL-6, which which are are involved involved in NO in NO and andIL-6 IL-6production, production, were were analyzed. analyzed. The effectThe effect of LPS of on LPS mRNA on mRNA expression expression was significantly was significantly suppressed suppressed following following treatment treatment with RW0117with RW0117 (Figure (Figure 4). These4). These data datasuggested suggested that thatRW0117 RW0117 possessed possessed anti-inflammatory anti-inflammatory ac- tivityactivity related related to toNO NO and and IL-6 IL-6 production production in in RAW264.7 RAW264.7 cells. cells. In In addition, addition, treatment treatment with with RW0117RW0117 in in low low concentration concentration (50 (50 μµg/mL)g/mL) showed showed more more effective effective than than beeswax beeswax alcohol. alcohol.

FigureFigure 4. 4. EffectEffect of of RW0117 RW0117 on on NO/IL-6 NO/IL-6 production production and and NO/IL-6 NO/IL-6 mRNA mRNA expression expression in LPS- in LPS-stimulatedstimulated RAW264.7 RAW264.7 cells; cells; (−): nontreated(−): nontreated group, group, (+): group (+): group treated treated with with500 ng/mL 500 ng/mL of LP ofS, LPS,Dexa: Dexa: dexamethasone-treated dexamethasone-treated group, group, beeswax: beeswax: beeswax beeswax al- cohol-treatedalcohol-treated group. group. Cells Cells were were treated treated with with either either dexame dexamethasone,thasone, beeswax, beeswax, or or RW0117 RW0117 for for 2 2h h and and then then incubated incubated with with LPSLPS for for 20 20 h. h. Results Results are are expressed as mean ± SDSD of of triplicate triplicate experiments. experiments. Statistical Statistical significance significance was was determined determined using using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. * p < 0.005 vs. LPS treated group. one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. * p < 0.005 vs. LPS treated group. 3.4. Analysis of the Effect of RW0117 on MAPK and NF-kB Signaling Pathways 3.4. Analysis of the Effect of RW0117 on MAPK and NF-kB Signaling Pathways To analyze the intracellular signal pathways involved in anti-inflammatory activity To analyze the intracellular signal pathways involved in anti-inflammatory activity of RW0117 in RAW264.7 cells, we examined LPS related signaling pathways proteins. of RW0117 in RAW264.7 cells, we examined LPS related signaling pathways proteins. First, phosphorylation of MAPK, a signaling protein representative of ERK, JNK, and p38, First, phosphorylation of MAPK, a signaling protein representative of ERK, JNK, and p38, activated by LPS, was confirmed by western blotting. As shown in Figure 5, LPS treatment activated by LPS, was confirmed by western blotting. As shown in Figure5, LPS treatment strongly induced ERK, JNK, and p38 phosphorylation in RAW264.7 cells. However, ERK and JNK phosphorylation was inhibited by treatment of RW0117 at a concentration of 100 Plants 2021, 10, x FOR PEER REVIEW 8 of 13

μg/mL and 50 μg/mL. A strong phosphorylation inhibitory activity, similar to that of the positive controls (Dexa. or beeswax alcohol) was observed in the group treated with 100 μg/mL of RW0117. However, it did not affect the phosphorylation of p38. In addition, phosphorylation of the transcription factor c-Jun, which is activated by the phosphoryla- tion of MAPK, was examined. The phosphorylation of c-Jun was strongly induced by treatment with LPS, and it was confirmed that this phosphorylation was inhibited by treatment with 100 μg/mL of RW0117 (Figure 5). Next, the NF-κB pathway, an inflammatory pathway activated by LPS, was analyzed. When cells are in normal state, IκBα protein exists in the cytoplasm as a complex with p65 Plants 2021, 10, 332 8 of 13 and p50. However, when stimulated by LPS, IκBα undergoes phosphorylation followed by decomposition. Simultaneously, phosphorylation of p65 occurs and the phosphory- lated protein is transported to the nucleus, where it acts as a transcription factor. As shown stronglyin Figure induced 5, phosphorylation ERK, JNK, andof p65 p38 was phosphorylation confirmed when in RAW264.7the cells were cells. treated However, with ERKLPS. andIn addition, JNK phosphorylation phosphorylation was of inhibitedp65 was slig byhtly treatment suppressed of RW0117 when treated at a concentration with beeswax of 100alcohol.µg/mL LPS-induced and 50 µg/mL. phosphorylation A strong phosphorylation was also slightly inhibitory suppressed activity, by similar treatment to that with of theRW0117. positive However, controls dexamethasone (Dexa. or beeswax did not alcohol) inhibit was p65 observed RW0117. in Moreover, the group phosphoryla- treated with 100tionµ ofg/mL iNOS of and RW0117. COX-2 However, proteins itwas did strongly not affect inhibited the phosphorylation by RW0117 (Figure of p38. 5). In Based addition, on phosphorylationthese results, it was of the predicted transcription that RW0117 factor c-Jun, inhibits which the is production activated by of the inflammatory phosphorylation fac- oftors MAPK, such as was NO, examined. IL-6, and The COX-2 phosphorylation by inhibiting of the c-Jun JNK/ERK was strongly pathway induced and activity by treatment of the withtranscription LPS, and factor it was c-Jun confirmed rather that than this inhibiting phosphorylation phosphorylation was inhibited of the by NF- treatmentκB pathway with 100(Figureµg/mL 5). of RW0117 (Figure5).

Figure 5. Effect of RW0117 onon thethe phosphorylationphosphorylation ofof severalseveral signalingsignaling proteinsproteins inin LPS-stimulatedLPS-stimulated RAW264.7 cells; (−): DMSO (0.05 %)-treated group, Beeswax: beeswax alcohol-treated group, Dexa.: RAW264.7 cells; (−): DMSO (0.05 %)-treated group, Beeswax: beeswax alcohol-treated group, Dexa.: dexamethasone-treated group, (+): group treated with 500 ng/mL of LPS. Whole cell-lysates were dexamethasone-treated group, (+): group treated with 500 ng/mL of LPS. Whole cell-lysates were immunoblotted with the indicated specific-antibodies. β-Actin served as an internal loading control. immunoblotted with the indicated specific-antibodies. β-Actin served as an internal loading control. 3.5. Analysis of Nuclear Translocation of NF-kB p65 by RW0117 in RAW264.7 Cells Next, the NF-κB pathway, an inflammatory pathway activated by LPS, was analyzed. WhenWhen cells areRAW264.7 in normal cells state, were Iκ Btreatedα protein with exists LPS, inphosphorylation the cytoplasm as of a p65 complex and its with nuclear p65 andtranslocation p50. However, were whenobserved. stimulated As shown by LPS, in IFigureκBα undergoes 6, analysis phosphorylation via fluorescence followed staining by decomposition.demonstrated that Simultaneously, nuclear translocation phosphorylation of p65 stained of p65 with occurs fluorescein-5-isothiocyanate and the phosphorylated protein(FITC) occurs is transported due to treatmen to the nucleus,t with LPS. where However, it acts aswhen a transcription cells were treate factor.d with As shown beeswax in Figurealcohol5 ,and phosphorylation RW0117 for 2 h of and p65 then was treated confirmed with when LPS, it the was cells confirmed were treated that the with nuclear LPS. Intranslocation addition, phosphorylation of p65 was suppressed of p65 wascompared slightly to suppressed that observed when in the treated group with treated beeswax with alcohol. LPS-induced phosphorylation was also slightly suppressed by treatment with RW0117. However, dexamethasone did not inhibit p65 RW0117. Moreover, phosphory- lation of iNOS and COX-2 proteins was strongly inhibited by RW0117 (Figure5). Based on these results, it was predicted that RW0117 inhibits the production of inflammatory factors such as NO, IL-6, and COX-2 by inhibiting the JNK/ERK pathway and activity of the transcription factor c-Jun rather than inhibiting phosphorylation of the NF-κB pathway (Figure5).

3.5. Analysis of Nuclear Translocation of NF-kB p65 by RW0117 in RAW264.7 Cells When RAW264.7 cells were treated with LPS, phosphorylation of p65 and its nuclear translocation were observed. As shown in Figure6, analysis via fluorescence staining demonstrated that nuclear translocation of p65 stained with fluorescein-5-isothiocyanate Plants 2021, 10, 332 9 of 13

(FITC) occurs due to treatment with LPS. However, when cells were treated with beeswax

Plants 2021, 10, x FOR PEER REVIEW alcohol and RW0117 for 2 h and then treated with LPS, it was confirmed that the nuclear9 of 13 translocation of p65 was suppressed compared to that observed in the group treated LPSwith alone. LPS alone.These Theseresults results show that show RW0 that117 RW0117 weakly weakly inhibits inhibits the LPS-induced the LPS-induced NF-κB NF- sig-κB nalingsignaling pathway, pathway, and beeswax and beeswax alcohol alcohol shows shows stronger stronger inhibition inhibition than RW0117. than RW0117. This result This isresult consistent is consistent with the with inhibition the inhibition of p65 phosphorylation of p65 phosphorylation as shown as shownin Figure in 5. Figure 5.

Figure 6. RW0117 inhibits LPS-induced p65 nuclear translocation in RAW264.7 cells. RAW264.7 Figure 6. RW0117 inhibits LPS-induced p65 nuclear translocation in RAW264.7 cells. RAW264.7 cells were seeded onto 0.1% gelatin-coated coverslips for overnight. After that beeswax alcohol cells were seeded onto 0.1% gelatin-coated coverslips for overnight. After that beeswax alcohol (100 μg/mL) or RW0117 (100 μg/mL, 50 μg/mL) treated for 2 h, then stimulated with LPS (1 µ µ µ µ μ(100g/mL)g/mL) for 1 h. or RAW264.7 RW0117 (100 cells g/mL,were fixed 50 g/mL)and immunostained treated for 2 h, with then anti-p65-FITC stimulated with antibody LPS (1 andg/mL) thefor nucleus 1 h. RAW264.7 was counterstained cells were fixed with and DAPI. immunostained The fluorescence with anti-p65-FITCimages were acquired antibody using and the ZEN nucleus 2.3 confocalwas counterstained imaging software. with DAPI.LPS was The used fluorescence as a positive images control were for activation acquired usingof RAW264.7 ZEN 2.3 cells. confocal imaging software. LPS was used as a positive control for activation of RAW264.7 cells. 3.6. Protective Effects of RW0117 in Rats with HCl/EtOH-Induced Gastric Ulcers 3.6. Protective Effects of RW0117 in Rats with HCl/EtOH-Induced Gastric Ulcers Ethanol is a well-known damaging agent to the gastric mucosa, and excessive ethanol Ethanol is a well-known damaging agent to the gastric mucosa, and excessive ethanol ingestion, serving as the main inducer of gastric ulcers in humans, causes acute gastric ingestion, serving as the main inducer of gastric ulcers in humans, causes acute gastric mucosal damage. After inducing stomach ulcers with HCl/EtOH, the rats were sacrificed mucosal damage. After inducing stomach ulcers with HCl/EtOH, the rats were sacrificed and their stomachs were excised. The amount of mucin was analyzed using periodic acid- and their stomachs were excised. The amount of mucin was analyzed using periodic Schiff staining [17]. Compared to that in the normal group (G1), the amount of acidic mu- acid-Schiff staining [17]. Compared to that in the normal group (G1), the amount of acidic cin (blue) in the group with gastric ulceration (G2) was significantly reduced. However, it mucin (blue) in the group with gastric ulceration (G2) was significantly reduced. However, increased in the positive control group treated with omeprazole + HCl/EtOH (G3) and in it increased in the positive control group treated with omeprazole + HCl/EtOH (G3) and the beeswax alcohol 100 + HCl/EtOH (G4) group, compared to that in the G2 group. Sim- in the beeswax alcohol 100 + HCl/EtOH (G4) group, compared to that in the G2 group. ilar results were obtained in the RW0117-treated groups G5 (RW0117: 25 mg/kg) and G6 Similar results were obtained in the RW0117-treated groups G5 (RW0117: 25 mg/kg) and (RW0117:G6 (RW0117: 50 mg/kg); 50 mg/kg); the stomachs the stomachs of rats of rats treated treated with with low-dose low-dose RW0117 RW0117 (G5) (G5) showed showed similarsimilar amounts amounts of of acidic acidic mucin mucin compared compared to to those those in in the the positive positive control control group, group, and and the the amountamount of of mucin mucin in in the the high-dose high-dose group group (G6) (G6) was was significantly significantly increased increased (Figure (Figure 7A).7A). Similar to the results obtained with alcian blue staining, the amount of acidic/neutral mucin (magenta) significantly decreased in the group with gastric ulceration (G2) com- pared to the normal group (G1). The omeprazole-treated positive control group (G3) showed a significant increase in acidic/neutral mucin, and the beeswax alcohol-treated group (G4) showed a slight increase. The mucin level in the gastric mucosa of the low- dose RW0117 group (G5) was similar to that of the positive control group. The amount of Plants 2021, 10, x FOR PEER REVIEW 10 of 13 Plants 2021, 10, 332 10 of 13 mucin in the gastric mucosa of the high-dose RW0117 group (G6) showed a similar sig- nificant increase to that of the gastric mucosa of the G1 group (Figure 7B).

Figure 7. Alcian/periodic acid-Schiff staining of HCl-EtOH induced ulceration in rat stomach. Upper pictures (G1–G6): Figure 7. Alcian/periodic acid-Schiff staining of HCl-EtOH induced ulceration in rat stomach. Upper Alcian staining for acidic mucin, Lower pictures (G1-G6): Periodic acid-Schiff staining for acidic/neutral mucin; G1: Nor- pictures (G1–G6): Alcian staining for acidic mucin, Lower pictures (G1-G6): Periodic acid-Schiff mal group, G2: HCl/EtOH treated group, G3: omeprazole, G4: beeswax alcohol 100 mg/kg G5: low-dose of RW0117 (25 mg/kg), G6: high-dosestaining for of acidic/neutralRW0117 (50 mg/kg) mucin; (scale G1: Normalbar = 50 group,μm). (A G2:) Alcian HCl/EtOH blue staining treated of group, HCl-EtOH G3: omeprazole, induced ulceration in rat stomach.G4: (B beeswax) Periodic alcohol acid-Schiff 100 staining mg/kg G5:of HCl-Et low-doseOH ofinduced RW0117 ulceration (25 mg/kg), in rat G6: stomach. high-dose of RW0117 (50 mg/kg) (scale bar = 50 µm). (A) Alcian blue staining of HCl-EtOH induced ulceration in rat stomach. (B) Periodic3.7. Analysis acid-Schiff of the staining Gene Expressions of HCl-EtOH of MUC1 induced, MUC5AC, ulceration in and rat MUC6 stomach. in Gastric Ulcer in Rats

Similar to theTo results verify obtained the results with obtained alcian blue after staining, staining, the the amount expression of acidic/neutral of genes, such as MUC1, mucin (magenta)MUC5AC, significantly and MUC6, decreased in the in thestomach group tissue with gastricwas measured. ulceration The (G2) HCl-EtOH-induced compared gas- to the normaltric group ulcer (G1). group The (control) omeprazole-treated showed a decrease positive in control the expression group (G3) of showed MUC1, aMUC5A, and significant increaseMUC6 ingenes acidic/neutral compared to mucin, the normal and the group beeswax (normal). alcohol-treated In the control group groups (G4) treated with showed a slight increase. The mucin level in the gastric mucosa of the low-dose RW0117 group (G5) was similar to that of the positive control group. The amount of mucin in the Plants 2021, 10, 332 11 of 13

gastric mucosa of the high-dose RW0117 group (G6) showed a similar significant increase to that of the gastric mucosa of the G1 group (Figure7B).

3.7. Analysis of the Gene Expressions of MUC1, MUC5AC, and MUC6 in Gastric Ulcer in Rats To verify the results obtained after staining, the expression of genes, such as MUC1, MUC5AC, and MUC6, in the stomach tissue was measured. The HCl-EtOH-induced Plants 2021, 10, x FORgastric PEER REVIEW ulcer group (control) showed a decrease in the expression of MUC1, MUC5A, 11 of 13 and MUC6 genes compared to the normal group (normal). In the control groups treated omeprazole, beeswax alcohol 100 mg/kg, and low-dose of RW0117 (25 mg/kg), the de- with omeprazole, beeswax alcohol 100 mg/kg, and low-dose of RW0117 (25 mg/kg), the crease in gene expression was not reversed. However, the expression of MUC5AC in the decrease in gene expression was not reversed. However, the expression of MUC5AC in positive control group was recovered to the same level as that of the normal group the positive control group was recovered to the same level as that of the normal group (omeprazole, beeswax alcohol). The group treated with high-dose RW0117 (50 mg/kg) (omeprazole, beeswax alcohol). The group treated with high-dose RW0117 (50 mg/kg) showed an increase in the expression level of MUC1 compared to the control group, and showed an increase in the expression level of MUC1 compared to the control group, the gene expression of MUC5AC and MUC6 increased significantly. These results, to- and the gene expression of MUC5AC and MUC6 increased significantly. These results, together withgether the histopathology with the histopathology results described results above, descr showibed above, that a differentshow that pathway a different pathway may be involvedmay in be the involved protective in actionthe protective of beeswax action alcohol of beeswax 100 and alcohol RW0117 100 on and the gastricRW0117 on the gas- mucosa othertric than mucosa increasing other the than expression increasing of the genes expre encodingssion of mucin. genes encoding In addition, mucin. the In addition, group treatedthe with group high-dose treated RW0117 with high-dose showed RW0117 a significant showed increase a significant in mucin, increase as per thein mucin, as per histopathologythe and histopathology molecular biology and molecular findings; biology therefore, findings; it was inferred therefore, that it itwas is more inferred that it is directly involvedmore in directly the protection involved of in the the gastric protection mucosa of throughthe gastric the mucosa secretion through of mucus the secretion of from the gastricmucus mucosa from than the thatgastric observed mucosa in than the other that groups.observed In in addition, the other it isgroups. expected In addition, it is that further studiesexpected are that needed further on studies the fact are that needed omeparzole, on the fa a drugct that that omeparzole, acts as a protona drug that acts as a pump inhibitor,proton did notpump affect inhibitor, muc1 and did muc6 not affect mRNA muc1 expression. and muc6 (Figure mRNA8). expression. (Figure 8).

Figure 8. Analysis of mRNA expression of Muc1, Muc5ac, Muc6 in ulcer-induced rat stomach. Figure 8. AnalysisUntreated: of mRNA nontreated expression group, of HCl/EtOH: Muc1, Muc5ac, HCl/EtOH-treated Muc6 in ulcer-induced group, Omeprazole: rat stomach.omeprazole Untreated: nontreated + group, HCl/EtOH: HCl/EtOH-treated group, Omeprazole: omeprazole + HCl/EtOH-treated group, beeswax alcohol: bees- HCl/EtOH-treated group, beeswax alcohol: beeswax alcohol 100 mg/kg + HCl/EtOH treated group, wax alcohol 100 mg/kg + HCl/EtOH treated group, RW0117 25 mg/kg: RW0117 25 mg/kg + HCl/EtOH-treated group, RW0117 25 mg/kg: RW0117 25 mg/kg + HCl/EtOH-treated group, RW0117 50 mg/kg: RW0117 RW0117 50 mg/kg: RW0117 50 mg/kg + HCl/EtOH-treated group. Statistical significance was determined using one-way analysis of variance50 mg/kg (ANOVA) + HCl/EtOH-treated followed by group.Tukey’s Statistical post hoc significancetest. # p < 0.05 was vs. determined control group. using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. # p < 0.05 vs. control group. 4. Conclusions4. Conclusions Medicinal herbsMedicinal have recently herbs attractedhave recently attention attracted as health attention beneficial as foods health and beneficial source foods and materials forsource drug development. materials for drug Previous development. studies demonstrated Previous studies that demonstratedA. argyi have that var- A. argyi have ious physiologicalvarious functions physiological such asfunctions antioxidant, such antitumor,as antioxidant, anti-inflammatory, antitumor, anti-inflammatory, antico- anti- coagulant, antiosteoporotic activities, and immunomodulation. These functions corre- lated with flavones, organic acid, terpens, polysaccharides, and coumarins isolated from A. argyi [29–33]. In this study, we confirmed RW0117 showed anti-inflammatory activity such as in- hibition of NO/IL-6 production and iNOS/COX-2 protein expression mediated by sup- pression of ERK-JNK/c-Jun signal pathways in RAW264.7 cells. In addition, in vivo results indicated that treatment with high doses of RW0117 (50 mg/kg) helped recover certain amounts of acidic and neutral mucin and the expression levels of genes encoding mucin in rat models of acute gastric ulcer using HCl/ethanol. These results indicated that Plants 2021, 10, 332 12 of 13

agulant, antiosteoporotic activities, and immunomodulation. These functions correlated with flavones, organic acid, terpens, polysaccharides, and coumarins isolated from A. argyi [29–33]. In this study, we confirmed RW0117 showed anti-inflammatory activity such as inhibi- tion of NO/IL-6 production and iNOS/COX-2 protein expression mediated by suppression of ERK-JNK/c-Jun signal pathways in RAW264.7 cells. In addition, in vivo results indicated that treatment with high doses of RW0117 (50 mg/kg) helped recover certain amounts of acidic and neutral mucin and the expression levels of genes encoding mucin in rat models of acute gastric ulcer using HCl/ethanol. These results indicated that RW0117 can protect HCl/ethanol-induced rat from gastric mucosal injury through inhibiting inflammatory response. Furthermore, it can be predicted that eupatilin, jaceosidin, and hispidulin in RW0117 will be involved in the activity. Collectively, these results indicated that RW0117 (A. argyi extract) has strong anti- inflammatory and antigastric ulcer activity and can be developed as a therapeutic agent or dietary supplement for stomach ulcers in future.

Supplementary Materials: The following are available online at https://www.mdpi.com/2223-774 7/10/2/332/s1, Table S1: DNA sequencing methods and results of Artemisia argyi. Author Contributions: M.-S.S. and K.S.K. conceived and designed the experiments; M.-S.S., J.L., and S.H.P. performed the experiments; M.-S.S. and K.S.K. analyzed the data; and J.W.L., S.H.P., I.K.L., J.A.C., J.S.L., and K.S.K. contributed reagents/materials/analysis tools; M.-S.S. wrote the paper. All authors have read and agreed to the published version of the manuscript. Funding: Richwood Pharmaceuticals Co. Ltd. Data Availability Statement: Not applicable. Conflicts of Interest: The authors declare no conflict of interest.

References 1. Zedler, S.; Faist, E. The impact of endogenous triggers on trauma-associated inflammation. Curr. Opin. Crit. Care 2006, 12, 595–601. [CrossRef][PubMed] 2. Isomäki, P.; Punnonen, J. Pro- and anti-inflammatory cytokines in rheumatoid arthritis. Ann. Med. 1997, 29, 499–507. [CrossRef] [PubMed] 3. Vane, J.R.; Mitchell, J.A.; Appleton, I.; Tomlinson, A.; Bishop-Bailey, D.; Croxtall, J.; Willoughby, D.A. Inducible isoforms of cyclooxygenase and nitric-oxide synthase in inflammation. Proc. Natl. Acad. Sci. USA 1994, 91, 2046–2050. [CrossRef] 4. Higuchi, M.; Higashi, N.; Taki, H.; Osawa, T. Cytolytic mechanisms of activated macrophages. Tumor necrosis factor and Larginine-dependent mechanisms act synergistically as the major cytolytic mechanisms of activated macrophages. J. Immunol. 1990, 144, 1425–1431. [PubMed] 5. Nathan, C. Nitric oxide as a secretory product of mammalian cells. FASEB J. 1992, 6, 3051–3064. [CrossRef][PubMed] 6. Guzik, T.J.; Korbut, R.; Adamek-Guzik, T. Nitric oxide and superoxide in inflammation and immune regulation. J. Physiol. Pharmacol. 2003, 54, 469–487. 7. Franke, A.; Teyssen, S.; Singer, M.V. Alcohol-related diseases of the esophagus and stomach. Dig. Dis. 2005, 23, 204–213. [CrossRef] 8. Marshal, B.J.; Waren, J.R. Unidentified curved baciliin the stomach of patients with gastritis and peptic ulceration. Lancet 1984, 1, 131–1315. 9. Honda, S.; Fujioka, T.; Tokieda, M.; Satoh, R.; Nishizono, A.; Nasu, M. Development of Helicobacter pylori-induced gastric carcinoma in Mongolian gerbils. Cancer Res. 1998, 58, 425–4259. 10. Goel, R.K.; Bhattacharya, S.K. Gastroduodenal mucosal defence and mucosal protective agents. Indian J. Exp. Biol. 1991, 29, 701–714. 11. DeVault, K.R.; Talley, N.J. Insights into the future of gastric acid suppression. Nat. Rev. Gastroenterol. Hepatol. 2009, 6, 524–532. [CrossRef][PubMed] 12. Li, C.Y.; Xu, H.D.; Zhao, B.T.; Chang, H.I.; Rhee, H.I. Gastroprotective effect of cyanidin 3-glucoside on ethanol-induced gastric lesions in rats. Alcohol 2008, 42, 683–687. [CrossRef] 13. Zhang, F.; Wang, L.; Wang, J.J.; Luo, P.F.; Wang, X.T.; Xia, Z.F. The caspase-1 inhibitor AC-YVAD-CMK attenuates acute gastric injury in mice: Involvement of silencing NLRP3 inflammasome activities. Sci. Rep. 2016, 6, 24166. [CrossRef] 14. Li, W.; Huang, H.; Niu, X.; Fan, T.; Mu, Q.; Li, H. Protective effect of tetrahydrocoptisine against ethanol-induced gastric ulcer in mice. Toxicol. Appl. Pharmacol. 2013, 272, 21–29. [CrossRef] Plants 2021, 10, 332 13 of 13

15. Vinay, S.C.; Pushpesh, K.M.; Rakesh, M.; Dharmani, P.; Gautam, P. Allophylus serratus: A plant with potential anti-ulcerogenic activity. J. Ethnopharmacol. 2005, 99, 361–366. 16. Sai, K.B.; Radha, K.L.; Gowrinath, R.M. Anti-ulcer effect of Aloe vera in non-steroidal anti-inflammatory drug induced peptic ulcers in rats. Afr. J. Pharm. Pharmacol. 2011, 5, 1867–1871. 17. Li, Q.; Hu, X.; Xuan, Y.; Ying, J.; Fei, Y.; Rong, J.; Liu, Z. Kaempferol protects ethanol-induced gastric ulcers in mice via pro-inflammatory cytokines and NO. Acta. Biochim. Biophys. Sin. 2018, 50, 246–253. [CrossRef] 18. Yun, C.; Jung, Y.; Chun, W.; Yang, B.; Ryu, J.; Lim, C.; Cho, S.I. Anti-inflammatory effects of Artemisia extract in mice with contact dermatitis in vitro and in vivo. Mediat. Inflamm. 2016.[CrossRef] 19. Shin, N.-R.; Ryu, H.-W.; Ko, J.-W.; Park, S.-H.; Yuk, H.-J.; Kim, H.-J. Artemisia argyi attenuates airway inflammation in ovalbumin- induced asthmatic animals. J. Ethnopharmacol. 2017, 209, 108–115. [CrossRef] 20. Zimmermann-Klemd, A.M.; Reinhardt, J.K.; Morath, A.; Schamel, W.W.; Steinberger, P.; Leitner, J.; Gründemann, C. Immunosup- pressive Activity of Artemisia argyi Extract and Isolated Compounds. Front. Pharmacol. 2020, 11, 402. [CrossRef] 21. Kim, S.M.; Lee, S.J.; Venkatarame Gowda Saralamma, V.; Ha, S.E.; Vetrivel, P.; Desta, K.T.; Kim, G.S. Polyphenol mixture of a native Korean variety of Artemisia argyi H. (Seomae ) and its anti-inflammatory effects. Int. J. Mol. Med. 2019, 44, 1741–1752. [CrossRef] 22. Choi, E.J.; Lee, S.; Chae, J.R.; Lee, H.S.; Jun, C.D.; Kim, S.H. Eupatilin inhibits lipopolysaccharide-induced expression of inflammatory mediators in macrophages. Life Sci. 2011, 88, 1121–1126. [CrossRef] 23. Min, S.W.; Kim, N.J.; Baek, N.I.; Kim, D.H. Inhibitory effect of eupatilin and jaceosidin isolated from Artemisia princeps on carrageenan-induced inflammation in mice. J. Ethnopharmacol. 2009, 125, 497–500. [CrossRef][PubMed] 24. Kim, D.E.; Min, K.J.; Kim, M.J.; Kim, S.H.; Kwon, T.K. Hispidulin inhibits mast cell-mediated allergic inflammation through down-regulation of histamine release and inflammatory cytokines. Molecules 2019, 24, 2131. [CrossRef] 25. Shin, M.S.; Song, J.H.; Choi, P.; Lee, J.H.; Kim, S.Y.; Shin, K.S.; Kang, K.S. Stimulation of innate immune function by panax ginseng after heat processing. J. Agric. Food Chem. 2018, 66, 4652–4659. [CrossRef] 26. Shin, M.S.; Kim, S.B.; Lee, J.; Choi, H.S.; Park, J.; Park, J.Y.; Kang, K.S. Beneficial effect of herbal formulation KM1608 on inflammatory bowel diseases: A preliminary experimental study. Molecules 2018, 23, 2068. [CrossRef] 27. Kwon, S.C.; Kim, J.H. Gastroprotective effects of irsogladine maleate on ethanol/hydrochloric acid induced gastric ulcers in mice. Korean J. Intern. Med. 2021, 36, 67–75. [CrossRef] 28. Rodríguez, I.; Illnait, J.; Terry, H.; Mas, R.; Fernández, L.; Fernández, J.C.; Cruz, Y. Effects of Abexol (beeswax alcohols) on gastrointestinal symptoms in middle-aged and older subjects. Rev. CENIC Cienc. Biol. 2009, 40, 147–154. 29. Song, X.; Wen, X.; He, J.; Zhao, H.; Li, S.; Wang, M. Phytochemical components and biological activities of Artemisia Argyi. J. Funct. Foods 2019, 52, 648–662. [CrossRef] 30. Kim, J.K.; Shin, E.C.; Lim, H.J.; Choi, S.J.; Kim, C.R.; Suh, S.H.; Shin, D.H. Characterization of nutritional composition, antioxida- tive capacity, and sensory attributes of Seomae mugwort, a native Korean variety of Artemisia argyi H. Lev. & Vaniot. J. Anal. Methods Chem. 2015, 2015, 1–9. 31. Seo, J.M.; Kang, H.M.; Son, K.H.; Kim, J.H.; Lee, C.W.; Kim, H.M.; Kwon, B.M. Antitumor activity of flavones isolated from Artemisia argyi. Planta Med. 2003, 69, 218–222. [CrossRef][PubMed] 32. Lv, J.L.; Li, Z.Z.; Zhang, L.B. Two new flavonoids from Artemisia argyi with their anticoagulation activities. Nat. Prod. Res. 2018, 32, 632–639. [CrossRef][PubMed] 33. Zhang, P.; Shi, B.; Li, T.; Xu, Y.; Jin, X.; Guo, X.; Yan, S. Immunomodulatory effect of Artemisia argyi polysaccharide on peripheral blood leucocyte of broiler chickens. J. Anim. Physiol. Anim. Nutr. 2018, 102, 939–946. [CrossRef][PubMed]