**1. Introduction**

Alcohol consumption is a recognized risk factor for human health. The most common diseases include infectious diseases, gastric ulcer, cancer, diabetes, and liver and pancreas disease caused by alcohol consumption either partially or entirely [1]. The pathogenesis of gastric ulcer is complicated and multifactorial; it is usually caused by an acute imbalance between gastric mucosal integrity and mucosal immunity [2]. Usually, ethanol is absorbed through the intestinal wall and metabolized in the liver in different ways: oxidation by alcohol dehydrogenase (ADH), cytochrome P450 2E1 (CYP2E1), and catalase enzymes. All the processes intensify to form acetaldehyde and then acetate by aldehyde dehydrogenase (ALDH). Alcohol metabolism with ADH enhances the generation of reduced forms of nicotinamide adenine dinucleotide (NADH), but production of CYP2E1 continues to produce free radical. Acetaldehyde and free radicals combine with cell compounds and disturb cell physiology [3]. Consequently, oxidative stress plays a crucial role in the pathogenesis of alcoholic tissue damage and increases lipid peroxidation, which injures capillary endothelial cells and increases cellular permeability [4] that are involved in the DNA damage of gastric mucosal epithelial cells [5]. Although the complete mechanism of alcohol-induced gastric mucosal damage has not been fully disclosed, evidence shows that oxidative stress and neutrophil infiltration are associated with the development of acute gastritis [6,7].

Lipopolysaccharide (LPS), a bacterial endotoxin, is commonly used as an inducer of the macrophage cell lineage, acting through Toll-like receptor 4 (TLR4), which activates the mitogen-activated protein kinases (MAPKs) signaling cascades and the pathway that triggers nuclear factor kappa B (NF-κB) [8,9]. MAPKs are the important signaling pathway and play a crucial regulatory role in both adapted and innate immune response [10]. Ethanol-induced oxidative stress stimulates the release of reactive oxygen species (ROS). ROS are recognized as the second messenger to initiate the redox-sensitive signal-transduction pathway with MAPK cascade and are linked with downstream transcription factor: NF-κB [11]. ROS mediate stimulation of inhibitor kappa B (IκB) kinase, which induces proteasomal breakdown of IκBα and activates NF-κB. NF-κB is a transcription factor that binds to κ-β motifs in the promoters of target genes and triggers transcription of inflammatory cytokines and chemokines [12].

The therapeutic and biological activities of indigenous plants and their active compounds have potential importance for their capability to manage and treat many inflammatory and immunomodulatory diseases [13]. *Rabdosia inflexa* (RI), a perennial shrub, is a member of the lamiaceae family, which is cosmopolitan and cultivated throughout Northeast China, the Korean peninsula, and Japan. In South Korea, RI, locally known as "sanbakha", has been used as folk medicine for treating gastrointestinal inflammation and pain. Previously, RI and its active compounds such as inflexin and inflexinol have been reported for pancreatitis and anti-cancer effect [14–16]. Based on its traditional uses and biological activities, the study investigated its anti-inflammatory and gastroprotective activity and its possible molecular mechanisms in both RAW 264.7 cells and HCl/EtOH-induced gastric ulcer in mice.

#### **2. Results**

#### *2.1. Analysis of Total Phenolic and Flavonoid Contents of Rabdosia inflexa (RI)*

Phenolic and flavonoid contents are the secondary metabolites of a plant, having a wide range of biological activities and usually antioxidant properties. The total phenolic and flavonoid content of RI were investigated and presented in Table 1. The total phenolic and flavonoid content of RI were 143.288 ± 1.68 mg/g gallic acid and 256.301 ± 1.40 mg/g rutin equivalent, respectively.


**Table 1.** Total phenolic and flavonoid content of *Rabdosia inflexa* (RI).

Note: Gallic acid and rutin were used as standards. Results are expressed in milligrams of gallic acid equivalent per gram of extract sample (mg GAE/g) and mg of rutin equivalent per gram of extract sample (mg RU/g).

#### *2.2. Effect of RI on Viability and Morphology of RAW 264.7 Cells*

The present study measured the anti-inflammatory ability of RI extract using RAW 264.7 cells on LPS-induced inflammation using MTT assay. To investigate the cytotoxicity and cell viability of RI, RAW 264.7 cells were treated with different concentrations of RI (50, 100, 200, 400, and 800 μg/mL) for 24 h. Among the concentrations, RI (800 μg/mL) significantly reduced the cell viability (Figure 1a). However, the cell viability did not significantly alter after co-treatment with LPS (0.5 μg/mL) and RI (100, 200, and 400 μg/mL) for 24 h (Figure 1b). As shown in Figure 1c, LPS markedly induced morphological changes of RAW 264.7 cells after 24 h of treatment, which was consequently improved by the treatment with RI. Thus, results proposed that RI has not affected the viability and morphology of RAW 264.7 cells and it could be due to the anti-inflammatory effect of RI.

**Figure 1.** Protective role of *Rabdosia inflexa* (RI) on: (**a**) cytotoxicity; (**b**) cell viability; and (**c**) morphological alteration in RAW 264.7 cells were measured by MTT assay. Cells were pretreated with various concentration of RI (100, 200, and 400 μg/mL) for 1 h, followed by co-treatment with RI and LPS (0.5 μg/mL) for another 24 h. Cell morphology was visualized by optical microscopy (scale bar 200 μm). \* *p* < 0.05 when compared with the control. Data are expressed as mean ± SEM of three independent experiments.
