Gastroprotective effects of a traditional ginseng–ginger formulation in a stress-induced gastritis mouse model

Gastroprotective effects of a traditional ginseng–ginger formulation in a stress-induced gastritis mouse model | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Gastroprotective effects of a traditional ginseng–ginger formulation in a stress-induced gastritis mouse model Jung Su-Ryun, Park Yu-Kyoung, Cha Hye-Na, Kwon Han Ol, Kim Jong Han, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8630439/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract This study investigated the gastroprotective effects of a traditional ginseng–ginger formulation using a water immersion and restraint stress (WIRS)-induced gastritis mouse model. Among the tested mixing ratios, the 3:1 (ginseng:ginger) formulation was the most effective in reducing gastric hemorrhagic lesions. Oral administration of the selected formulation for seven days significantly attenuated stress-induced increases in inflammatory markers, including interleukin-1β, inducible nitric oxide synthase, and cyclooxygenase-2. In addition, the formulation reduced lipid peroxidation while enhancing glutathione peroxidase activity, indicating improved antioxidant defense capacity. Notably, these protective effects were independent of changes in gene expression related to gastric acid secretion. Taken together, the findings suggest that a traditional ginseng–ginger formulation may contribute to maintaining gastric health under stress conditions through modulation of inflammation- and oxidative stress–associated responses, supporting its potential relevance as a dietary strategy for maintaining gastric health under stress conditions. Ginseng Ginger Traditional food formulation Stress-induced gastritis Oxidative stress Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Gastritis, an inflammatory disorder of the gastric mucosa, affects nearly half of the global population and represents a significant gastrointestinal health burden. Its development is associated with multiple factors including Helicobacter pylori infection, alcohol consumption, smoking, nonsteroidal anti-inflammatory drug (NSAID) use, and psychological stress ( 1 ). Although improvements in sanitation and socioeconomic conditions have contributed to a reduction in H. pylori-associated gastritis in Korea ( 2 ), stress-related gastric injury and NSAID-associated gastric ulcers remain important clinical problems, particularly in aging populations. Pharmacological therapies such as histamine H2 receptor antagonists and proton pump inhibitors are widely used to treat gastric inflammation and ulceration. However, long-term administration of these drugs may cause undesirable adverse effects. For instance, cimetidine has been associated with endocrine-related side effects including gynecomastia and galactorrhea ( 3 ), while proton pump inhibitors such as omeprazole and lansoprazole may induce gastrointestinal symptoms including nausea, abdominal pain, constipation, and diarrhea ( 4 ). These limitations have encouraged the search for alternative preventive strategies capable of protecting gastric mucosa with fewer adverse effects. Stress-induced gastric injury is primarily mediated by inflammatory activation and oxidative damage in gastric tissues. In experimental models such as water immersion and restraint stress (WIRS), gastric mucosal injury is characterized by hemorrhagic lesions accompanied by increased expression of inflammatory mediators including interleukin-1β (IL-1β), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2), as well as excessive generation of reactive oxygen species (ROS) and lipid peroxidation ( 5 – 10 ). Because these inflammatory and oxidative pathways play central roles in the pathogenesis of stress-induced gastric damage, therapeutic strategies capable of modulating both processes simultaneously may provide effective gastroprotective effects. Importantly, stress-induced gastric injury involves complex interactions among gastric mucosal integrity, inflammatory responses, oxidative stress, and systemic physiological regulation, and therefore evaluation of potential gastroprotective interventions requires an integrated in vivo experimental model. Stress-induced gastritis models such as water immersion and restraint stress (WIRS) are widely used experimental systems for evaluating gastroprotective agents that target inflammatory and oxidative pathways involved in gastric mucosal injury. Korean Red Ginseng ( Panax ginseng Meyer ) has long been used in Asian traditional medicine and has been reported to exert anti-inflammatory and cytoprotective effects in gastric tissues by regulating cytokine production and oxidative stress-related signaling pathways ( 11 – 15 ). Ginger ( Zingiber officinale Roscoe ), a widely consumed culinary herb, also possesses well-documented anti-inflammatory and antioxidant activities ( 16 – 18 ). Experimental studies have demonstrated that ginger and its bioactive constituents, including 6-gingerol and 6-shogaol, suppress COX-2 and NF-κB signaling pathways and attenuate oxidative gastric injury in cellular and animal models ( 19 – 21 ). Although Korean Red Ginseng and ginger each exhibit gastroprotective properties, their combined effects on stress-induced gastric mucosal injury remain largely unexplored. Importantly, these two botanical ingredients appear to act through partially distinct but complementary biological mechanisms. Korean Red Ginseng primarily suppresses inflammatory cytokine production and modulates immune signaling pathways ( 13 – 15 ), whereas ginger inhibits COX-2/NF-κB activation and enhances antioxidant defense systems that counteract oxidative stress ( 17 – 20 ). Because stress-related gastric injury results from the interaction of inflammatory activation and oxidative imbalance ( 8 – 10 ), combining these two ingredients may provide broader protective effects by concurrently targeting multiple pathogenic pathways involved in gastric mucosal damage. Moreover, increasing evidence suggests that botanical combinations containing multiple bioactive compounds may exhibit enhanced therapeutic efficacy through synergistic or complementary interactions among phytochemicals, allowing simultaneous modulation of multiple molecular targets involved in complex inflammatory diseases ( 22 , 23 ). Such multi-component strategies are increasingly explored in phytopharmacology because single-target interventions are often insufficient to control multifactorial pathological processes. However, despite the well-documented individual gastroprotective properties of ginseng and ginger, the efficacy of a ginseng–ginger formulation in stress-induced gastric injury has not been systematically investigated. Therefore, the present study aimed to investigate the gastroprotective effects of a ginseng–ginger formulation in a water immersion and restraint stress (WIRS)-induced gastritis mouse model. In particular, we examined whether the combined formulation could attenuate gastric mucosal damage by modulating inflammatory mediators and oxidative stress-related pathways in gastric tissue. Materials and Methods Animals Male C57BL/6J mice (8 weeks old) were purchased from Geo Research (Daegu, Republic of Korea) and maintained under specific pathogen-free conditions at the animal facility of Yeungnam University College of Medicine. After a one-week acclimation period, mice were randomly assigned to experimental groups. Mice were housed in pairs at 21 °C with ad libitum access to food (Harlan Teklad, USA) and water. All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Yeungnam University College of Medicine (approval number: YUMC-AEC2021-010). Preparation of Korean Red Ginseng and ginger extracts Korean Red Ginseng (Panax ginseng Meyer, Araliaceae) extract powder was obtained from the Korean Ginseng Corporation (Gwacheon, Republic of Korea). Ginger extract powder, sourced from India (Umalaximi Organics PVT. LTD, batch no. UOOSZO2017-0520), was prepared via four sequential extractions with 70% aqueous ethanol. For ratio screening, red ginseng and ginger extracts were mixed at 1:3, 1:1, and 3:1 (w/w). The term “PZ” refers to the 3:1 (red ginseng:ginger) formulation, which was selected based on ratio screening and used for dose determination and subsequent experiments. Standardization of Korean Red Ginseng and ginger extracts Ginsenoside HPLC analysis : Analytical-grade reagents and HPLC-grade acetonitrile and methanol (Merck) were used. Standards of ginsenoside Rg1, Rb1, and 20(S)-Rg3 were obtained from Chromadex. For sample preparation, 0.5 g of red ginseng extract powder was extracted with 10 mL methanol via ultrasonic treatment (60 Hz, 30 min), centrifuged (30,000 x g , 15 min), and filtered (pore 0.2 µm). Analysis was performed using a Waters ACQUITY UPLC system (BEH C18 column, 1.7 µm, 50 × 2.1 mm) at 30 °C, employing a gradient of water and acetonitrile at 0.6 mL/min. Injection volume was 2 µL, and detection was at 203 nm using a photodiode array detector. Gingerol HPLC analysis : 6-Gingerol and 6-shogaol standards were obtained from Chromadex (Irvine, CA, USA). PZ powder (0.2 g) was extracted with 10 mL of 70% methanol for 60 minutes, centrifuged at 3,000 × g for 10 minutes (Legend Mach 1.6R, Thermo Fisher Scientific), and filtered through a 0.2 µm Acrodisc® filter (Sigma). Analysis was performed using HPLC-PDA (Waters) with a Zorbax eclipse XDB-C18 column at 45 °C. The mobile phase composed of 0.01% phosphoric acid in water (A), acetonitrile (B) with a gradient: 15–45% B (0–15 min), 50% B (21 min), 70% B (23 min), 90% B (26 min), then returning to 45% B (30 min). Injection volume was 20 µL, flow rate 1.0 mL/min, and detection at 280 nm. Water immersion and restraint stress (WIRS)-induced gastritis The water immersion and restraint stress (WIRS) model is widely used to induce stress-related gastritis in mice, characterized by gastric mucosal erosion with inflammation and oxidative stress (5-7). Animals were maintained under fasting conditions for 24 hours before the experiment. The test compound was administered orally 1 hour before stress exposure. Gastric injury was induced by restraining mice individually in cages submerged in 25 °C water for 6 hours (Picture 1). Picture 1. Representative device used for WIRS induction Experimental protocols The experimental procedure is summarized in Figure 1. The study comprised three phases, with group assignments and sample sizes detailed in Table 1. Step 1. Determination of Pg and Zo mixing ratio : Pg (KGC12 Pg, Panax ginseng) and Zo (KGC12 Zo, Zingiber officinale) were administered orally once daily for 7 days, either individually or in mixtures at ratios of 1:3, 1:1, and 3:1. After a 24-h fasting period, mice received the final dose 1 hour prior to WIRS-induced gastritis. Gastric tissues were collected 6 hours post-WIRS for analysis. Step 2. PZ (3:1) dosage determination: The 3:1 Pg:Zo mixture (PZ) was administered orally once daily at 100, 200, and 300 mg/kg for 7 days. Procedures were identical to those in step 1. Step 3. Verification of the effect of 300 mg/kg PZ : Mice received 300 mg/kg PZ or vehicle orally once daily for 7 days. Subsequent procedures followed the protocol described in step 1. Tissue extraction Immediately after WIRS, animals were deeply anesthetized with avertin (2,2,2-tribromoethanol, 250 mg/kg, i.p.), and adequate anesthesia was confirmed by the absence of reflex responses. Under deep anesthesia, blood samples were collected and the stomach was excised for analysis. Following tissue collection, animals were euthanized by cervical dislocation to ensure death, in accordance with institutional and national guidelines for animal care and use. Tissues were rapidly frozen and stored at −80 °C until further analysis. Blood samples were anticoagulated with heparin, centrifuged (1500 × g, 15 min), and plasma was collected and stored at −80 °C for further analysis. Gastric mucosal hemorrhage area The excised stomach was incised along the greater curvature, spread on a Styrofoam plate, and fixed. After washing with saline, the stomach was photographed, and the areas of gastric bleeding and total stomach surface were quantified using ImageJ (NIH, Bethesda, MD, USA). The bleeding area was calculated using the following formula: Hemorrhagic area (%) = (hemorrhagic area/total area) × 100 Analysis Blood factor: IL-1β concentrations were analyzed using the Mouse IL-1β ELISA mLB00C, following the manufacturer's analytical guidelines (R&D Systems, MN, USA). Malondialdehyde (MDA) assay: MDA levels in gastric tissue were measured using a TBARS kit (Cayman Chemical, #10009055). Tissue homogenates were centrifuged, and 100 µL of supernatant was assayed. Protein concentration was measured by Bradford assay for normalization. Glutathione peroxidase (GPx) activity: The activity of GPx was measured using a Cayman Chemical kit (#703102) via a glutathione reductase–coupled assay with cumene hydroperoxide. Absorbance at 340 nm was recorded for 5 min. One unit corresponds to oxidation of 1.0 nmol NADPH per minute at 25 °C. Western blotting: Stomach tissues were homogenized in lysis buffer containing protease inhibitors, subjected to freeze–thaw cycles, and centrifuged. Protein concentration was determined using the Bradford assay. Equal amounts of protein were separated by SDS–PAGE and transferred onto PVDF membranes. The membranes were incubated with primary antibodies against iNOS (#13120, Cell Signaling Technology, USA), COX-2 (#12282, Cell Signaling Technology, USA), IL-1β (sc-1251, Santa Cruz Biotechnology, USA), GPx (LF-PA0248, AbFrontier, Korea), MDA (ab27642, Abcam, UK), and GAPDH (sc-32233, Santa Cruz Biotechnology, USA), followed by incubation with horseradish peroxidase-conjugated secondary antibodies. Protein bands were visualized using the LAS-4000 imaging system (Fujifilm, Japan) and quantified using ImageJ software (NIH, USA). Protein expression levels were normalized to GAPDH. Real-Time Reverse Transcription PCR: Total RNA was separated from the gastric tissue using TRI reagent (Sigma, USA), followed by sonication, phase separation, isopropanol precipitation, ethanol washing, and resuspension in RNase-free water. RNA concentration was measured by NanoDrop (Thermo Scientific, USA). From 1 µg RNA, cDNA was produced using the cDNA Reverse Transcription Kit (Applied Biosystems, USA). Expression levels were normalized to 36B4. Primer sequences (Bioneer, Korea) were: H2R: 5′-TCCTAAGCGACCCGGTACAGC-3′/5′-ATGGAGACTGAGGCACTGCTGG-3′ CCK2R: 5′-GATGGCTGCTACGTGCAACT-3′/5′-CGCACCACCCGCTTCTTAG-3′ M3R: 5′-CACAATAACAGTACAACCTCGCC-3′/5′-GCCAGGATGCCCGTTAAGAAA-3′ 36B4: 5′-GATGCCCAGGGAAGACAG-3′/5′-CAATGAAGCATTTTGGATAATCA-3′ Statistical analysis The GraphPad Prism version 5 (GraphPad Software, USA) was employed to analyze the experimental data. All values are given as mean ± SEM. Differences between the groups were evaluated by one-way ANOVA. To further analyze group differences, post hoc comparisons among groups were performed using Tukey’s test. Statistical significance was less than 0.05. Results Chromatographic characterization of PZ UPLC and HPLC analyses identified three major ginsenosides (Rg1, Rb1, and Rg3s) and two ginger-derived compounds (6-gingerol and 6-shogaol) in PZ (Fig. 2 ). The total content of Rg1, Rb1, and Rg3s was 5.95 mg/g, and that of 6-gingerol and 6-shogaol was 11.63 mg/g, confirming the presence of active constituents from both Korean Red Ginseng and ginger extracts. Protective effects of PZ (3:1) against WIRS-induced gastritis Oral administration of Korean Red Ginseng (Pg), ginger (Zo), or their mixture (PZ, 3:1) at 300 mg/kg for 7 days did not affect food intake or body weight (Fig. 3 A, B). After 6 h of water-immersion restraint stress (WIRS), all treatment groups showed a reduction in gastric hemorrhage compared with the control, whereas stomach weight remained unchanged (Fig. 3 C, D). The PZ (3:1) group exhibited a significant decrease in the hemorrhagic area (Fig. 3 E), indicating a gastroprotective effect. A dose–response study further demonstrated that PZ (3:1) reduced gastric bleeding in a concentration-dependent manner, and the 300 mg/kg dose exerted the strongest protective effect (Fig. 4 A, B). Therefore, this concentration was used for subsequent analyses. Effects of PZ (3:1) on inflammatory markers The anti-inflammatory potential of PZ (3:1) was evaluated by measuring the expression of inflammatory mediators in gastric tissue. WIRS markedly increased IL-1β, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) expression (Fig. 4 C–F), which are key enzymes involved in the inflammatory response. Pretreatment with PZ (3:1) significantly attenuated the WIRS-induced upregulation of these mediators, suggesting that the mixture suppresses stress-induced inflammation. As IL-1β promotes the expression of iNOS and COX-2, this coordinated down-regulation indicates that PZ (3:1) exerts broad anti-inflammatory effects in the gastric mucosa. Antioxidant effects of PZ (3:1) Oxidative stress contributes to the pathogenesis of stress-induced gastritis through excessive production of reactive oxygen species (ROS). In this study, PZ (3:1) significantly decreased malondialdehyde (MDA) levels, a marker of lipid peroxidation, and increased glutathione peroxidase (GPx) activity in gastric tissues (Fig. 5 A, B). Although protein levels of MDA and GPx1 were not significantly changed (Fig. 5 C–E), these findings suggest that PZ enhances antioxidant enzyme activity rather than protein abundance. Thus, the PZ mixture likely mitigates oxidative injury by reinforcing the antioxidant defense system, particularly the GPx pathway. Effect of PZ (3:1) on gastric acid secretion To determine whether the protective action of PZ (3:1) involves modulation of gastric acid secretion, mRNA expression levels of muscarinic acetylcholine receptor M3 (M3R), histamine H2 receptor (H2R), and cholecystokinin 2 receptor (CCK2R) were examined. PZ treatment did not significantly affect the expression of these genes (Fig. 5 F–H), indicating that the gastroprotective effect is independent of acid secretion control. Discussion The stomach is highly sensitive to both internal and external stressors and is therefore prone to a wide range of disorders, from mild indigestion to severe diseases such as gastric ulcer and gastric cancer, which are associated with high morbidity and mortality. Acute gastritis is characterized by the activation of inflammatory and oxidative pathways, including increased expression of cytokines such as IL-6, IL-1β, and TNF-α ( 24 ), infiltration of neutrophils and monocytes ( 25 ), and the production of reactive oxygen species (ROS) that disrupt the oxidative balance ( 26 , 27 ). In particular, COX-2 and inducible nitric oxide synthase (iNOS) are key enzymes involved in oxidative stress-related gastric inflammation ( 8 – 10 ). IL-1β is a pivotal mediator of acute and chronic inflammation, particularly in Helicobacter pylori infection ( 28 ). COX-2 contributes to gastric mucosal hemorrhage by promoting inflammatory cell recruitment and the production of prostaglandins associated with edema, pain, and fever ( 29 ), while iNOS expression in gastric epithelial cells mediates oxidative and inflammatory responses to infection or stress ( 30 ). Although pharmacological agents such as proton pump inhibitors and H2 receptor blockers are commonly used to treat gastritis, their long-term administration often causes adverse effects. Therefore, safe, food-derived bioactive compounds are being explored as alternative prophylactic options. Korean Red Ginseng and ginger are well-known traditional ingredients with reported gastroprotective effects. Korean Red Ginseng has been shown to reduce gastritis severity by suppressing IL-1β and iNOS expression in H. pylori-infected mucosa ( 13 – 15 ), while ginger extract inhibits COX-2 and NF-κB activation and decreases ethanol-induced oxidative injury ( 20 , 21 ). In the present study, a combination of Korean Red Ginseng and ginger extracts (PZ mixture) was evaluated in a water-immersion restraint stress (WIRS) model, which induces acute gastric injury through oxidative stress, inflammation, and mucosal erosion ( 6 , 31 , 32 ). Through a three-phase experimental design, the 3:1 ratio of Korean Red Ginseng to ginger at 300 mg/kg was identified as the most effective formulation for preventing WIRS-induced gastric damage. At this dose, PZ significantly reduced hemorrhagic lesions and markedly suppressed the protein expression of IL-1β, iNOS, and COX-2 in gastric tissue, indicating a reduction in stress-associated inflammatory markers. Since IL-1β promotes iNOS and COX-2 expression, the concurrent down-regulation of these mediators suggests that the observed gastroprotective effects are associated with attenuation of stress-induced inflammatory responses. WIRS-induced gastric injury is also closely linked to lipid peroxidation and oxidative imbalance caused by excessive ROS generation. In this study, the ginseng–ginger formulation significantly reduced malondialdehyde levels and increased glutathione peroxidase activity without altering protein expression levels. This pattern suggests an enhancement of functional antioxidant capacity rather than enzyme overexpression, which has been reported as a characteristic response to food-derived bioactive compounds under acute stress conditions. From a dietary perspective, the present findings suggest that a traditional ginseng–ginger formulation may contribute to gastric resilience under stress conditions. Rather than acting through modulation of gastric acid secretion, the observed effects appear to be associated with attenuation of inflammatory responses and enhancement of antioxidant defense capacity. Given the long history of dietary use of ginseng and ginger in Asian food culture, this formulation may represent a traditional dietary approach for supporting gastric health during periods of stress. Future studies will be required to determine whether the observed gastroprotective effects involve pharmacological synergy between the bioactive compounds present in ginseng and ginger. Declarations Conflict of interest This study was supported by Korea Ginseng Corporation (KGC). The authors declare that the funding organization had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Ethics statement The experiments in this study were approved by the Institutional Animal Care and Use Committee (IACUC) of Yeungnam University College of Medicine (YUMC-AEC2021-010) and were conducted in full compliance with ARRIVE guidelines and institutional ethical standards. Competing Interests This study was supported by Korea Ginseng Corporation (KGC). The authors declare that the funding organization had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Funding This research was supported by the Korea Ginseng Corporation (KGC-MD 20–221). Author Contribution S.Y.P. designed the study. Y.K.P. and H.N.C., H.O.K., J.H.K., B.S.B. performed the experiments. S.R.J. and Y.K.P. wrote the manuscript. All authors approved the final version. Acknowledgement The authors would like to express their sincere gratitude to the Korea Ginseng Corporation (KGC) for financial support (Project No. KGC-MD 20-221) and for providing the KGC12PZ samples used in this study. The authors also thank the Laboratory Animal Research Center at Yeungnam University College of Medicine for technical assistance with animal experiments. We are grateful to our colleagues in the Department of Pharmacology for valuable discussions and constructive comments during the course of this work. Data Availability The datasets generated and/or analyzed during the current study are available from the corresponding author upon reasonable request. References Thorsen K, Søreide JA, Kvaløy JT, Glomsaker T, Søreide K. Epidemiology of perforated peptic ulcer: age- and gender-adjusted analysis of incidence and mortality. World J Gastroenterol. 2013;19(3):347–54. Chauhan AK, Kang SC. Therapeutic potential and mechanism of thymol action against ethanol-induced gastric mucosal injury in rat model. Alcohol. 2015;49(7):739–45. Kurata JH, Nogawa AN. Meta-analysis of risk factors for peptic ulcer. Nonsteroidal antiinflammatory drugs, Helicobacter pylori, and smoking. J Clin Gastroenterol. 1997;24(1):2–17. Yeomans ND, Hawkey CJ, Brailsford W, Naesdal J. Gastroduodenal toxicity of low-dose acetylsalicylic acid: a comparison with non-steroidal anti-inflammatory drugs. Curr Med Res Opin. 2009;25(11):2785–93. Ock CY, Hong KS, Choi KS, Chung MH, Kim Y, Kim JH, et al. A novel approach for stress-induced gastritis based on paradoxical anti-oxidative and anti-inflammatory action of exogenous 8-hydroxydeoxyguanosine. Biochem Pharmacol. 2011;81(1):111–22. Xu B, Cui X, Xu K, Lin L, Zhou Y. Effect of water immersion restraint stress on gastric mucosa in rats with removed salivary glands. Int J Clin Exp Pathol. 2020;13(11):2853–9. Fan F, Yang M, Geng X, Ma X, Sun H. Effects of Restraint Water-Immersion Stress-Induced Gastric Mucosal Damage on Astrocytes and Neurons in the Nucleus Raphe Magnus of Rats via the ERK1/2 Signaling Pathway. Neurochem Res. 2019;44(8):1841–50. Yisireyili M, Alimujiang A, Aili A, Li Y, Yisireyili S, Abudureyimu K. Chronic Restraint Stress Induces Gastric Mucosal Inflammation with Enhanced Oxidative Stress in a Murine Model. Psychol Res Behav Manag. 2020;13:383–93. Brzozowski T, Konturek PC, Mierzwa M, Drozdowicz D, Bielanski W, Kwiecien S, et al. Effect of probiotics and triple eradication therapy on the cyclooxygenase (COX)-2 expression, apoptosis, and functional gastric mucosal impairment in Helicobacter pylori-infected Mongolian gerbils. Helicobacter. 2006;11(1):10–20. Fu S, Ramanujam KS, Wong A, Fantry GT, Drachenberg CB, James SP, et al. Increased expression and cellular localization of inducible nitric oxide synthase and cyclooxygenase 2 in Helicobacter pylori gastritis. Gastroenterology. 1999;116(6):1319–29. Wang Y, Guan WX, Zhou Y, Zhang XY, Zhao HJ. Red ginseng polysaccharide promotes ferroptosis in gastric cancer cells by inhibiting PI3K/Akt pathway through down-regulation of AQP3. Cancer Biol Ther. 2024;25(1):2284849. Kim JK, Shin KK, Kim H, Hong YH, Choi W, Kwak YS, et al. Korean Red Ginseng exerts anti-inflammatory and autophagy-promoting activities in aged mice. J Ginseng Res. 2021;45(6):717–25. Kang H, Lim JW, Kim H. Inhibitory effect of Korean Red Ginseng extract on DNA damage response and apoptosis in Helicobacter pylori-infected gastric epithelial cells. J Ginseng Res. 2020;44(1):79–85. Bae M, Jang S, Lim JW, Kang J, Bak EJ, Cha JH, et al. Protective effect of Korean Red Ginseng extract against Helicobacter pylori-induced gastric inflammation in Mongolian gerbils. J Ginseng Res. 2014;38(1):8–15. Kim HS, Lim JW, Kim H. Korean Red Ginseng Extract Inhibits IL-8 Expression via Nrf2 Activation in Helicobacter pylori-Infected Gastric Epithelial Cells. Nutrients. 2022;14(5). Nikkhah Bodagh M, Maleki I, Hekmatdoost A. Ginger in gastrointestinal disorders: A systematic review of clinical trials. Food Sci Nutr. 2019;7(1):96–108. Haniadka R, Saldanha E, Sunita V, Palatty PL, Fayad R, Baliga MS. A review of the gastroprotective effects of ginger (Zingiber officinale Roscoe). Food Funct. 2013;4(6):845–55. Abidi C, Rtibi K, Boutahiri S, Tounsi H, Abdellaoui A, Wahabi S, et al. Dose-dependent Action of Zingiber officinale on Colonic Dysmotility and Ex Vivo Spontaneous Intestinal Contraction Modulation. Dose Response. 2022;20(3):15593258221127556. Gaus K, Huang Y, Israel DA, Pendland SL, Adeniyi BA, Mahady GB. Standardized ginger (Zingiber officinale) extract reduces bacterial load and suppresses acute and chronic inflammation in Mongolian gerbils infected with cagAHelicobacter pylori. Pharm Biol. 2009;47(1):92–8. Song MY, Lee DY, Park SY, Seo SA, Hwang JS, Heo SH, et al. Steamed Ginger Extract Exerts Anti-inflammatory Effects in Helicobacter pylori-infected Gastric Epithelial Cells through Inhibition of NF-κB. J Cancer Prev. 2021;26(4):289–97. Song MY, Lee DY, Park SY, Seo SA, Hwang JS, Heo SH, et al. Erratum: Steamed Ginger Extract Exerts Anti-inflammatory Effects in Helicobacter pylori-infected Gastric Epithelial Cells through Inhibition of NF-κB. J Cancer Prev. 2022;27(1):77. Wagner H. Synergy research: approaching a new generation of phytopharmaceuticals. Fitoterapia. 2011;82(1):34–7. Williamson EM. Synergy and other interactions in phytomedicines. Phytomedicine. 2001;8(5):401–9. Wang W, Li Q, Yan X, Chen Z, Xie Y, Hu H, et al. Comparative study of raw and processed Vladimiriae Radix on pharmacokinetic and anti-acute gastritis effect through anti-oxidation and anti-inflammation. Phytomedicine. 2020;70:153224. Ren WK, Xu YF, Wei WH, Huang P, Lian DW, Fu LJ, et al. Effect of patchouli alcohol on Helicobacter pylori-induced neutrophil recruitment and activation. Int Immunopharmacol. 2019;68:7–16. Salim AS. Use of scavenging oxygen-derived free radicals to protect the rat against aspirin- and ethanol-induced erosive gastritis. J Pharm Sci. 1992;81(9):943–6. Al-Quraishy S, Othman MS, Dkhil MA, Abdel Moneim AE. Olive (Olea europaea) leaf methanolic extract prevents HCl/ethanol-induced gastritis in rats by attenuating inflammation and augmenting antioxidant enzyme activities. Biomed Pharmacother. 2017;91:338–49. Hong JB, Zuo W, Wang AJ, Lu NH. Helicobacter pylori Infection Synergistic with IL-1β Gene Polymorphisms Potentially Contributes to the Carcinogenesis of Gastric Cancer. Int J Med Sci. 2016;13(4):298–303. Laine L, Jensen DM. Management of patients with ulcer bleeding. Am J Gastroenterol. 2012;107(3):345–60. quiz 61. Barrachina MD, Panés J, Esplugues JV. Role of nitric oxide in gastrointestinal inflammatory and ulcerative diseases: perspective for drugs development. Curr Pharm Des. 2001;7(1):31–48. Ding H, Gao Y, Wang Y, Yao K, Wang G, Zhang J. The role of peripheral serotonin and norepinephrine in the gastroprotective effect against stress of duloxetine. Eur J Pharmacol. 2023;941:175499. Liu X, Yuan Z, Luo L, Wang T, Zhao F, Zhang J, et al. Protective role of fruits of Rosa odorata var. gigantea against WIRS-induced gastric mucosal injury in rats by modulating pathway related to inflammation, oxidative stress and apoptosis. Chin Herb Med. 2024;16(2):263–73. Tables Tables are available in the Supplementary Files section. Additional Declarations Competing interest reported. This study was supported by Korea Ginseng Corporation (KGC). The authors declare that the funding organization had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Supplementary Files Table.pptx Suppl.PZ.pptx PDFSupplementaryfileOriginaluncroppedwesternblots.pdf Cite Share Download PDF Status: Under Review Version 1 posted Reviewers invited by journal 20 Apr, 2026 Editor assigned by journal 14 Apr, 2026 Editor invited by journal 14 Apr, 2026 Submission checks completed at journal 07 Apr, 2026 First submitted to journal 07 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8630439","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":626357318,"identity":"640d2b47-a244-488d-9b76-f78e9f667cf2","order_by":0,"name":"Jung Su-Ryun","email":"","orcid":"","institution":"Yeungnam University","correspondingAuthor":false,"prefix":"","firstName":"Jung","middleName":"","lastName":"Su-Ryun","suffix":""},{"id":626357319,"identity":"f86d4f8e-195f-405e-a555-03ed109a346d","order_by":1,"name":"Park Yu-Kyoung","email":"","orcid":"","institution":"Yeungnam University","correspondingAuthor":false,"prefix":"","firstName":"Park","middleName":"","lastName":"Yu-Kyoung","suffix":""},{"id":626357321,"identity":"b97a5c5f-4de2-4d5a-8da1-1352d9d303d7","order_by":2,"name":"Cha Hye-Na","email":"","orcid":"","institution":"Yeungnam University","correspondingAuthor":false,"prefix":"","firstName":"Cha","middleName":"","lastName":"Hye-Na","suffix":""},{"id":626357323,"identity":"494bbe43-c216-4a1d-982e-3e7f86c72cd1","order_by":3,"name":"Kwon Han Ol","email":"","orcid":"","institution":"Korea Ginseng Corporation","correspondingAuthor":false,"prefix":"","firstName":"Kwon","middleName":"Han","lastName":"Ol","suffix":""},{"id":626357324,"identity":"41e50ef1-a9ec-4805-b578-f920175cdc32","order_by":4,"name":"Kim Jong Han","email":"","orcid":"","institution":"Korea Ginseng Corporation","correspondingAuthor":false,"prefix":"","firstName":"Kim","middleName":"Jong","lastName":"Han","suffix":""},{"id":626357325,"identity":"32adb2e8-bfea-4920-af0b-4b21e9fdd7a4","order_by":5,"name":"Bae Bong Seok","email":"","orcid":"","institution":"Korea Ginseng Corporation","correspondingAuthor":false,"prefix":"","firstName":"Bae","middleName":"Bong","lastName":"Seok","suffix":""},{"id":626357326,"identity":"e2e97adb-f4ff-4f64-9f99-b17343eb16d1","order_by":6,"name":"Park So-Young","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7UlEQVRIiWNgGAWjYNCCAzZybPzNB6A8NqK0pBnzSRxLIEnLocR5DDkGxGmRn5F78HPFmQPGbAxnvkkXtjHI8zewpX3Ap8XgRl6y5Jkbd+TYmHu3Sc9sYzCccYDt8Ay8WiRyDCQbPjwD2nJ2mzRvGwPjBgb2ZgIOyzH+2fDhcGIbQ84zkBZ7gloYbuSYSTbcAGthA2lJ3MDAdhivDoMz79IsG86kGbNJHDO25jknkTzjMFsyfoe15x6+2XDMRk6+v/nhbZ4yG9v+9jZj/A4TyIEzWSQYGICIGb8GBgb+M3AmM97oGAWjYBSMgpELALuGSQM01UQ0AAAAAElFTkSuQmCC","orcid":"","institution":"Yeungnam University","correspondingAuthor":true,"prefix":"","firstName":"Park","middleName":"","lastName":"So-Young","suffix":""}],"badges":[],"createdAt":"2026-01-18 09:53:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8630439/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8630439/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108051113,"identity":"a22ccccc-279c-49d2-bccd-f2828eff340f","added_by":"auto","created_at":"2026-04-28 21:32:42","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":37361,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic representation of the experimental design. The outline summarizes the preparation of Korean Red Ginseng (Pg), ginger (Zo), and their 3:1 mixture (PZ), oral administration for 7 days, and induction of gastritis by water immersion and restraint stress (WIRS).\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8630439/v1/2df006f2e43e235f8bbb2cf9.jpg"},{"id":108181788,"identity":"d95d8b47-6622-42f6-abdf-6c035a8e2209","added_by":"auto","created_at":"2026-04-30 08:58:55","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":40455,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative chromatograms of ginsenoside standards (Rg1, Rb1, and Rg3(S)) and ginger constituents (6-gingerol and 6-shogaol), along with KGC12 and KGC12zo powders. Rg1, Rb1, and Rg3(S) were identified as marker compounds of Korean Red Ginseng extract (KGC12), while 6-gingerol and 6-shogaol were identified as marker compounds of the ginger-containing extract (KGC12zo). (A) Chromatogram of three standard ginsenoside mixtures, (B) Chromatogram of KGC12 pg, (C) Chromatogram of two standard mixtures, (D) Chromatogram of KGC12\u003cem\u003ezo.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8630439/v1/1af1c9b514915a51160bf689.jpg"},{"id":108051118,"identity":"801148bb-7228-4c0c-af48-b10d8b6214e0","added_by":"auto","created_at":"2026-04-28 21:32:42","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":104459,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of 7-day oral administration of Korean Red Ginseng (Pg), ginger (Zo), and their 3:1 combination (PZ) on body weight and water immersion and restraint stress (WIRS)-induced gastric bleeding. (A) Body weight; (B) Food consumption (g/day); (C) Representative gastric images; (D) Stomach weights; (E) Hemorrhagic area (%). Values are expressed as mean ± SEM (n = 10). p \u0026lt; 0.05 vs. WIRS.\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8630439/v1/623ea9abc8ee21db46c349a7.jpg"},{"id":108051120,"identity":"97b68f2b-8bef-41ab-9a90-b90c6ca96d4e","added_by":"auto","created_at":"2026-04-28 21:32:42","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":119738,"visible":true,"origin":"","legend":"\u003cp\u003eDose-dependent inhibitory effects of PZ on WIRS-induced hemorrhagic gastritis and modulation of inflammatory factor expression in gastric tissue. (A) Morphological appearance of gastric mucosa; (B) Quantitative analysis of hemorrhagic area; (C) Representative Western blots; (D) Protein expression of IL-1β; (E) iNOS; and (F) COX-2. Values are expressed as mean ± SEM (n = 7). p \u0026lt; 0.05 vs. WIRS.\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8630439/v1/8f0286e1d11ef8945c70dc2b.jpg"},{"id":108051119,"identity":"2a19f88e-7604-4be0-a0bc-55c32f77850e","added_by":"auto","created_at":"2026-04-28 21:32:42","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":95319,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of PZ (3:1, 300 mg/kg) on oxidative stress-related markers and gastric mucosal protective gene expression in WIRS-induced gastritis. (A) Malondialdehyde (MDA) levels; (B) Glutathione peroxidase (GPx) activity; (C) Representative Western blots; (D) Quantification of MDA expression; (E) Quantification of GPx expression; (F) mRNA levels of muscarinic acetylcholine receptor M3 (M3R); (G) Cholecystokinin 2 receptor (CCK2R); and (H) Histamine H2 receptor (H2R). Values are expressed as mean ± SEM (n = 7). p \u0026lt; 0.05, **p \u0026lt; 0.0001 vs. distilled water (DW) control.\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8630439/v1/aac71db9a550fbd9367a1f43.jpg"},{"id":108183812,"identity":"18151526-9348-458b-a537-f7545a873746","added_by":"auto","created_at":"2026-04-30 09:02:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":615154,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8630439/v1/02ce4d89-221c-44f1-a50b-d213b0937c25.pdf"},{"id":108181357,"identity":"262b3115-486a-4541-8093-274ac638a6f0","added_by":"auto","created_at":"2026-04-30 08:58:34","extension":"pptx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":37127,"visible":true,"origin":"","legend":"","description":"","filename":"Table.pptx","url":"https://assets-eu.researchsquare.com/files/rs-8630439/v1/ee07d41ece76f15c700758b2.pptx"},{"id":108051115,"identity":"c793f17a-bf23-4fd2-968c-a313ce6dd95d","added_by":"auto","created_at":"2026-04-28 21:32:42","extension":"pptx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":3188618,"visible":true,"origin":"","legend":"","description":"","filename":"Suppl.PZ.pptx","url":"https://assets-eu.researchsquare.com/files/rs-8630439/v1/4a581dfb36c9020130aeefe6.pptx"},{"id":108181755,"identity":"55301d35-8ee6-4c51-90a1-6c20b7106ffa","added_by":"auto","created_at":"2026-04-30 08:58:53","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":254556,"visible":true,"origin":"","legend":"","description":"","filename":"PDFSupplementaryfileOriginaluncroppedwesternblots.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8630439/v1/fa64a56269b07fb067dda5f1.pdf"}],"financialInterests":"Competing interest reported. This study was supported by Korea Ginseng Corporation (KGC). The authors declare that the funding organization had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.","formattedTitle":"\u003cp\u003eGastroprotective effects of a traditional ginseng–ginger formulation in a stress-induced gastritis mouse model \u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eGastritis, an inflammatory disorder of the gastric mucosa, affects nearly half of the global population and represents a significant gastrointestinal health burden. Its development is associated with multiple factors including \u003cem\u003eHelicobacter pylori\u003c/em\u003e infection, alcohol consumption, smoking, nonsteroidal anti-inflammatory drug (NSAID) use, and psychological stress (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Although improvements in sanitation and socioeconomic conditions have contributed to a reduction in H. pylori-associated gastritis in Korea (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e), stress-related gastric injury and NSAID-associated gastric ulcers remain important clinical problems, particularly in aging populations.\u003c/p\u003e \u003cp\u003ePharmacological therapies such as histamine H2 receptor antagonists and proton pump inhibitors are widely used to treat gastric inflammation and ulceration. However, long-term administration of these drugs may cause undesirable adverse effects. For instance, cimetidine has been associated with endocrine-related side effects including gynecomastia and galactorrhea (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e), while proton pump inhibitors such as omeprazole and lansoprazole may induce gastrointestinal symptoms including nausea, abdominal pain, constipation, and diarrhea (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). These limitations have encouraged the search for alternative preventive strategies capable of protecting gastric mucosa with fewer adverse effects.\u003c/p\u003e \u003cp\u003eStress-induced gastric injury is primarily mediated by inflammatory activation and oxidative damage in gastric tissues. In experimental models such as water immersion and restraint stress (WIRS), gastric mucosal injury is characterized by hemorrhagic lesions accompanied by increased expression of inflammatory mediators including interleukin-1β (IL-1β), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2), as well as excessive generation of reactive oxygen species (ROS) and lipid peroxidation (\u003cspan additionalcitationids=\"CR6 CR7 CR8 CR9\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Because these inflammatory and oxidative pathways play central roles in the pathogenesis of stress-induced gastric damage, therapeutic strategies capable of modulating both processes simultaneously may provide effective gastroprotective effects. Importantly, stress-induced gastric injury involves complex interactions among gastric mucosal integrity, inflammatory responses, oxidative stress, and systemic physiological regulation, and therefore evaluation of potential gastroprotective interventions requires an integrated in vivo experimental model. Stress-induced gastritis models such as water immersion and restraint stress (WIRS) are widely used experimental systems for evaluating gastroprotective agents that target inflammatory and oxidative pathways involved in gastric mucosal injury.\u003c/p\u003e \u003cp\u003eKorean Red Ginseng (\u003cem\u003ePanax ginseng Meyer\u003c/em\u003e) has long been used in Asian traditional medicine and has been reported to exert anti-inflammatory and cytoprotective effects in gastric tissues by regulating cytokine production and oxidative stress-related signaling pathways (\u003cspan additionalcitationids=\"CR12 CR13 CR14\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Ginger (\u003cem\u003eZingiber officinale Roscoe\u003c/em\u003e), a widely consumed culinary herb, also possesses well-documented anti-inflammatory and antioxidant activities (\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Experimental studies have demonstrated that ginger and its bioactive constituents, including 6-gingerol and 6-shogaol, suppress COX-2 and NF-κB signaling pathways and attenuate oxidative gastric injury in cellular and animal models (\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAlthough Korean Red Ginseng and ginger each exhibit gastroprotective properties, their combined effects on stress-induced gastric mucosal injury remain largely unexplored. Importantly, these two botanical ingredients appear to act through partially distinct but complementary biological mechanisms. Korean Red Ginseng primarily suppresses inflammatory cytokine production and modulates immune signaling pathways (\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e), whereas ginger inhibits COX-2/NF-κB activation and enhances antioxidant defense systems that counteract oxidative stress (\u003cspan additionalcitationids=\"CR18 CR19\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Because stress-related gastric injury results from the interaction of inflammatory activation and oxidative imbalance (\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e), combining these two ingredients may provide broader protective effects by concurrently targeting multiple pathogenic pathways involved in gastric mucosal damage.\u003c/p\u003e \u003cp\u003eMoreover, increasing evidence suggests that botanical combinations containing multiple bioactive compounds may exhibit enhanced therapeutic efficacy through synergistic or complementary interactions among phytochemicals, allowing simultaneous modulation of multiple molecular targets involved in complex inflammatory diseases (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Such multi-component strategies are increasingly explored in phytopharmacology because single-target interventions are often insufficient to control multifactorial pathological processes.\u003c/p\u003e \u003cp\u003eHowever, despite the well-documented individual gastroprotective properties of ginseng and ginger, the efficacy of a ginseng\u0026ndash;ginger formulation in stress-induced gastric injury has not been systematically investigated. Therefore, the present study aimed to investigate the gastroprotective effects of a ginseng\u0026ndash;ginger formulation in a water immersion and restraint stress (WIRS)-induced gastritis mouse model. In particular, we examined whether the combined formulation could attenuate gastric mucosal damage by modulating inflammatory mediators and oxidative stress-related pathways in gastric tissue.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eAnimals\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMale C57BL/6J mice (8 weeks old) were purchased from Geo Research (Daegu, Republic of Korea) and maintained under specific pathogen-free conditions at the animal facility of Yeungnam University College of Medicine. After a one-week acclimation period, mice were randomly assigned to experimental groups. Mice were housed in pairs at 21 \u0026deg;C with ad libitum access to food (Harlan Teklad, USA) and water. All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Yeungnam University College of Medicine (approval number: YUMC-AEC2021-010).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eKorean Red\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Ginseng and ginger extracts\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKorean Red Ginseng (Panax ginseng Meyer, Araliaceae) extract powder was obtained from the Korean Ginseng Corporation (Gwacheon, Republic of Korea). Ginger extract powder, sourced from India (Umalaximi Organics PVT. LTD, batch no. UOOSZO2017-0520), was prepared via four sequential extractions with 70% aqueous ethanol. For ratio screening, red ginseng and ginger extracts were mixed at 1:3, 1:1, and 3:1 (w/w). The term \u0026ldquo;PZ\u0026rdquo; refers to the 3:1 (red ginseng:ginger) formulation, which was selected based on ratio screening and used for dose determination and subsequent experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStandardization of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eKorean Red\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Ginseng and ginger extracts\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eGinsenoside HPLC analysis\u003c/u\u003e: Analytical-grade reagents and HPLC-grade acetonitrile and methanol (Merck) were used. Standards of ginsenoside Rg1, Rb1, and 20(S)-Rg3 were obtained from Chromadex. For sample preparation, 0.5 g of red ginseng extract powder was extracted with 10 mL methanol via ultrasonic treatment (60 Hz, 30 min), centrifuged (30,000 x \u003cem\u003eg\u003c/em\u003e, 15 min), and filtered (pore 0.2 \u0026micro;m). Analysis was performed using a Waters ACQUITY UPLC system (BEH C18 column, 1.7 \u0026micro;m, 50 \u0026times; 2.1 mm) at 30 \u0026deg;C, employing a gradient of water and acetonitrile at 0.6 mL/min. Injection volume was 2 \u0026micro;L, and detection was at 203 nm using a photodiode array detector.\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eGingerol HPLC analysis\u003c/u\u003e: 6-Gingerol and 6-shogaol standards were obtained from Chromadex (Irvine, CA, USA). PZ powder (0.2 g) was extracted with 10 mL of 70% methanol for 60 minutes, centrifuged at 3,000 \u0026times; g for 10 minutes (Legend Mach 1.6R, Thermo Fisher Scientific), and filtered through a 0.2 \u0026micro;m Acrodisc\u0026reg; filter (Sigma). Analysis was performed using HPLC-PDA (Waters) with a Zorbax eclipse XDB-C18 column at 45 \u0026deg;C. The mobile phase composed of 0.01% phosphoric acid in water (A), acetonitrile (B) with a gradient: 15\u0026ndash;45% B (0\u0026ndash;15 min), 50% B (21 min), 70% B (23 min), 90% B (26 min), then returning to 45% B (30 min). Injection volume was 20 \u0026micro;L, flow rate 1.0 mL/min, and detection at 280 nm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWater\u0026nbsp;immersion\u0026nbsp;and restraint\u0026nbsp;stress\u0026nbsp;(WIRS)-induced\u0026nbsp;gastritis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe water immersion and restraint stress (WIRS) model is widely used to induce stress-related gastritis in mice, characterized by gastric mucosal erosion with inflammation and oxidative stress (5-7). \u0026nbsp;Animals were maintained under fasting conditions for 24 hours before the experiment. The test compound was administered orally 1 hour before stress exposure. Gastric injury was induced by restraining mice individually in cages submerged in 25 \u0026deg;C water for 6 hours (Picture 1).\u003c/p\u003e\n\u003cp\u003ePicture 1.\u0026nbsp;Representative device used for WIRS induction\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExperimental protocols\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experimental procedure is summarized in Figure 1. The study comprised three phases, with group assignments and sample sizes detailed in Table 1.\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eStep 1. Determination of Pg and Zo mixing ratio\u003c/u\u003e: Pg (KGC12 Pg, Panax ginseng) and Zo (KGC12 Zo, Zingiber officinale) were administered orally once daily for 7 days, either individually or in mixtures at ratios of 1:3, 1:1, and 3:1. After a 24-h fasting period, mice received the final dose 1 hour prior to WIRS-induced gastritis. Gastric tissues were collected 6 hours post-WIRS for analysis.\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eStep 2. PZ (3:1) dosage determination:\u0026nbsp;\u003c/u\u003eThe 3:1 Pg:Zo mixture (PZ) was administered orally once daily at 100, 200, and 300 mg/kg for 7 days. Procedures were identical to those in step 1.\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eStep 3. Verification of the effect of 300 mg/kg PZ\u003c/u\u003e: Mice received 300 mg/kg PZ or vehicle orally once daily for 7 days. Subsequent procedures followed the protocol described in step 1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTissue\u0026nbsp;extraction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImmediately after WIRS, animals were deeply anesthetized with avertin (2,2,2-tribromoethanol, 250 mg/kg, i.p.), and adequate anesthesia was confirmed by the absence of reflex responses. Under deep anesthesia, blood samples were collected and the stomach was excised for analysis. Following tissue collection, animals were euthanized by cervical dislocation to ensure death, in accordance with institutional and national guidelines for animal care and use. Tissues were rapidly frozen and stored at \u0026minus;80 \u0026deg;C until further analysis. Blood samples were anticoagulated with heparin, centrifuged (1500 \u0026times; g, 15 min), and plasma was collected and stored at \u0026minus;80 \u0026deg;C for further analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGastric mucosal\u0026nbsp;hemorrhage\u0026nbsp;area\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe excised stomach was incised along the greater curvature, spread on a Styrofoam plate, and fixed. After washing with saline, the stomach was photographed, and the areas of gastric bleeding and total stomach surface were quantified using ImageJ (NIH, Bethesda, MD, USA). The bleeding area was calculated using the following formula:\u003c/p\u003e\n\u003cp\u003eHemorrhagic area (%) = (hemorrhagic area/total area) \u0026times; 100\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBlood factor:\u0026nbsp;\u003c/strong\u003eIL-1\u0026beta; concentrations were analyzed using the Mouse IL-1\u0026beta; ELISA mLB00C, following the manufacturer\u0026apos;s analytical guidelines (R\u0026amp;D Systems, MN, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMalondialdehyde (MDA) assay:\u0026nbsp;\u003c/strong\u003eMDA levels in gastric tissue were measured using a TBARS kit (Cayman Chemical, #10009055). Tissue homogenates were centrifuged, and 100 \u0026micro;L of supernatant was assayed. Protein concentration was measured by Bradford assay for normalization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGlutathione peroxidase (GPx) activity:\u0026nbsp;\u003c/strong\u003eThe activity of GPx was measured using a Cayman Chemical kit (#703102) via a glutathione reductase\u0026ndash;coupled assay with cumene hydroperoxide. Absorbance at 340 nm was recorded for 5 min. One unit corresponds to oxidation of 1.0 nmol NADPH per minute at 25 \u0026deg;C.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blotting:\u0026nbsp;\u003c/strong\u003eStomach tissues were homogenized in lysis buffer containing protease inhibitors, subjected to freeze\u0026ndash;thaw cycles, and centrifuged. Protein concentration was determined using the Bradford assay. Equal amounts of protein were separated by SDS\u0026ndash;PAGE and transferred onto PVDF membranes. The membranes were incubated with primary antibodies against iNOS (#13120, Cell Signaling Technology, USA), COX-2 (#12282, Cell Signaling Technology, USA), IL-1\u0026beta; (sc-1251, Santa Cruz Biotechnology, USA), GPx (LF-PA0248, AbFrontier, Korea), MDA (ab27642, Abcam, UK), and GAPDH (sc-32233, Santa Cruz Biotechnology, USA), followed by incubation with horseradish peroxidase-conjugated secondary antibodies. Protein bands were visualized using the LAS-4000 imaging system (Fujifilm, Japan) and quantified using ImageJ software (NIH, USA). Protein expression levels were normalized to GAPDH.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReal-Time Reverse Transcription PCR:\u0026nbsp;\u003c/strong\u003eTotal RNA was separated from the gastric tissue using TRI reagent (Sigma, USA), followed by sonication, phase separation, isopropanol precipitation, ethanol washing, and resuspension in RNase-free water. RNA concentration was measured by NanoDrop (Thermo Scientific, USA). From 1 \u0026micro;g RNA, cDNA was produced using the cDNA Reverse Transcription Kit (Applied Biosystems, USA). Expression levels were normalized to 36B4. Primer sequences (Bioneer, Korea) were:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eH2R: 5\u0026prime;-TCCTAAGCGACCCGGTACAGC-3\u0026prime;/5\u0026prime;-ATGGAGACTGAGGCACTGCTGG-3\u0026prime;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCCK2R: 5\u0026prime;-GATGGCTGCTACGTGCAACT-3\u0026prime;/5\u0026prime;-CGCACCACCCGCTTCTTAG-3\u0026prime;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eM3R: 5\u0026prime;-CACAATAACAGTACAACCTCGCC-3\u0026prime;/5\u0026prime;-GCCAGGATGCCCGTTAAGAAA-3\u0026prime;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e36B4: 5\u0026prime;-GATGCCCAGGGAAGACAG-3\u0026prime;/5\u0026prime;-CAATGAAGCATTTTGGATAATCA-3\u0026prime;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical\u0026nbsp;analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe GraphPad Prism version 5 (GraphPad Software, USA)\u0026nbsp;was employed to analyze the experimental data. All values are given as mean \u0026plusmn; SEM. Differences between the groups were evaluated by one-way ANOVA. To further analyze group differences, post hoc comparisons among groups were performed using Tukey\u0026rsquo;s test. Statistical significance was less than 0.05.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eChromatographic characterization of PZ\u003c/h2\u003e \u003cp\u003eUPLC and HPLC analyses identified three major ginsenosides (Rg1, Rb1, and Rg3s) and two ginger-derived compounds (6-gingerol and 6-shogaol) in PZ (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The total content of Rg1, Rb1, and Rg3s was 5.95 mg/g, and that of 6-gingerol and 6-shogaol was 11.63 mg/g, confirming the presence of active constituents from both Korean Red Ginseng and ginger extracts.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eProtective effects of PZ (3:1) against WIRS-induced gastritis\u003c/h2\u003e \u003cp\u003eOral administration of Korean Red Ginseng (Pg), ginger (Zo), or their mixture (PZ, 3:1) at 300 mg/kg for 7 days did not affect food intake or body weight (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B). After 6 h of water-immersion restraint stress (WIRS), all treatment groups showed a reduction in gastric hemorrhage compared with the control, whereas stomach weight remained unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, D). The PZ (3:1) group exhibited a significant decrease in the hemorrhagic area (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE), indicating a gastroprotective effect. A dose\u0026ndash;response study further demonstrated that PZ (3:1) reduced gastric bleeding in a concentration-dependent manner, and the 300 mg/kg dose exerted the strongest protective effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B). Therefore, this concentration was used for subsequent analyses.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eEffects of PZ (3:1) on inflammatory markers\u003c/h2\u003e \u003cp\u003eThe anti-inflammatory potential of PZ (3:1) was evaluated by measuring the expression of inflammatory mediators in gastric tissue. WIRS markedly increased IL-1β, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC\u0026ndash;F), which are key enzymes involved in the inflammatory response. Pretreatment with PZ (3:1) significantly attenuated the WIRS-induced upregulation of these mediators, suggesting that the mixture suppresses stress-induced inflammation. As IL-1β promotes the expression of iNOS and COX-2, this coordinated down-regulation indicates that PZ (3:1) exerts broad anti-inflammatory effects in the gastric mucosa.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eAntioxidant effects of PZ (3:1)\u003c/h2\u003e \u003cp\u003eOxidative stress contributes to the pathogenesis of stress-induced gastritis through excessive production of reactive oxygen species (ROS). In this study, PZ (3:1) significantly decreased malondialdehyde (MDA) levels, a marker of lipid peroxidation, and increased glutathione peroxidase (GPx) activity in gastric tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B). Although protein levels of MDA and GPx1 were not significantly changed (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC\u0026ndash;E), these findings suggest that PZ enhances antioxidant enzyme activity rather than protein abundance. Thus, the PZ mixture likely mitigates oxidative injury by reinforcing the antioxidant defense system, particularly the GPx pathway.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eEffect of PZ (3:1) on gastric acid secretion\u003c/h2\u003e \u003cp\u003eTo determine whether the protective action of PZ (3:1) involves modulation of gastric acid secretion, mRNA expression levels of muscarinic acetylcholine receptor M3 (M3R), histamine H2 receptor (H2R), and cholecystokinin 2 receptor (CCK2R) were examined. PZ treatment did not significantly affect the expression of these genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF\u0026ndash;H), indicating that the gastroprotective effect is independent of acid secretion control.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe stomach is highly sensitive to both internal and external stressors and is therefore prone to a wide range of disorders, from mild indigestion to severe diseases such as gastric ulcer and gastric cancer, which are associated with high morbidity and mortality. Acute gastritis is characterized by the activation of inflammatory and oxidative pathways, including increased expression of cytokines such as IL-6, IL-1β, and TNF-α (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e), infiltration of neutrophils and monocytes (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e), and the production of reactive oxygen species (ROS) that disrupt the oxidative balance (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). In particular, COX-2 and inducible nitric oxide synthase (iNOS) are key enzymes involved in oxidative stress-related gastric inflammation (\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). IL-1β is a pivotal mediator of acute and chronic inflammation, particularly in Helicobacter pylori infection (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). COX-2 contributes to gastric mucosal hemorrhage by promoting inflammatory cell recruitment and the production of prostaglandins associated with edema, pain, and fever (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e), while iNOS expression in gastric epithelial cells mediates oxidative and inflammatory responses to infection or stress (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAlthough pharmacological agents such as proton pump inhibitors and H2 receptor blockers are commonly used to treat gastritis, their long-term administration often causes adverse effects. Therefore, safe, food-derived bioactive compounds are being explored as alternative prophylactic options. Korean Red Ginseng and ginger are well-known traditional ingredients with reported gastroprotective effects. Korean Red Ginseng has been shown to reduce gastritis severity by suppressing IL-1β and iNOS expression in H. pylori-infected mucosa (\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e), while ginger extract inhibits COX-2 and NF-κB activation and decreases ethanol-induced oxidative injury (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn the present study, a combination of Korean Red Ginseng and ginger extracts (PZ mixture) was evaluated in a water-immersion restraint stress (WIRS) model, which induces acute gastric injury through oxidative stress, inflammation, and mucosal erosion (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Through a three-phase experimental design, the 3:1 ratio of Korean Red Ginseng to ginger at 300 mg/kg was identified as the most effective formulation for preventing WIRS-induced gastric damage. At this dose, PZ significantly reduced hemorrhagic lesions and markedly suppressed the protein expression of IL-1β, iNOS, and COX-2 in gastric tissue, indicating a reduction in stress-associated inflammatory markers. Since IL-1β promotes iNOS and COX-2 expression, the concurrent down-regulation of these mediators suggests that the observed gastroprotective effects are associated with attenuation of stress-induced inflammatory responses.\u003c/p\u003e \u003cp\u003eWIRS-induced gastric injury is also closely linked to lipid peroxidation and oxidative imbalance caused by excessive ROS generation. In this study, the ginseng\u0026ndash;ginger formulation significantly reduced malondialdehyde levels and increased glutathione peroxidase activity without altering protein expression levels. This pattern suggests an enhancement of functional antioxidant capacity rather than enzyme overexpression, which has been reported as a characteristic response to food-derived bioactive compounds under acute stress conditions.\u003c/p\u003e \u003cp\u003eFrom a dietary perspective, the present findings suggest that a traditional ginseng\u0026ndash;ginger formulation may contribute to gastric resilience under stress conditions. Rather than acting through modulation of gastric acid secretion, the observed effects appear to be associated with attenuation of inflammatory responses and enhancement of antioxidant defense capacity. Given the long history of dietary use of ginseng and ginger in Asian food culture, this formulation may represent a traditional dietary approach for supporting gastric health during periods of stress. Future studies will be required to determine whether the observed gastroprotective effects involve pharmacological synergy between the bioactive compounds present in ginseng and ginger.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of interest\u003c/h2\u003e \u003cp\u003eThis study was supported by Korea Ginseng Corporation (KGC). The authors declare that the funding organization had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eEthics statement\u003c/h2\u003e \u003cp\u003e The experiments in this study were approved by the Institutional Animal Care and Use Committee (IACUC) of Yeungnam University College of Medicine (YUMC-AEC2021-010) and were conducted in full compliance with ARRIVE guidelines and institutional ethical standards.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003cp\u003eThis study was supported by Korea Ginseng Corporation (KGC). The authors declare that the funding organization had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis research was supported by the Korea Ginseng Corporation (KGC-MD 20\u0026ndash;221).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eS.Y.P. designed the study. Y.K.P. and H.N.C., H.O.K., J.H.K., B.S.B. performed the experiments. S.R.J. and Y.K.P. wrote the manuscript. All authors approved the final version.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors would like to express their sincere gratitude to the Korea Ginseng Corporation (KGC) for financial support (Project No. KGC-MD 20-221) and for providing the KGC12PZ samples used in this study. The authors also thank the Laboratory Animal Research Center at Yeungnam University College of Medicine for technical assistance with animal experiments. We are grateful to our colleagues in the Department of Pharmacology for valuable discussions and constructive comments during the course of this work.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets generated and/or analyzed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eThorsen K, S\u0026oslash;reide JA, Kval\u0026oslash;y JT, Glomsaker T, S\u0026oslash;reide K. Epidemiology of perforated peptic ulcer: age- and gender-adjusted analysis of incidence and mortality. World J Gastroenterol. 2013;19(3):347\u0026ndash;54.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChauhan AK, Kang SC. Therapeutic potential and mechanism of thymol action against ethanol-induced gastric mucosal injury in rat model. Alcohol. 2015;49(7):739\u0026ndash;45.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKurata JH, Nogawa AN. Meta-analysis of risk factors for peptic ulcer. Nonsteroidal antiinflammatory drugs, Helicobacter pylori, and smoking. J Clin Gastroenterol. 1997;24(1):2\u0026ndash;17.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYeomans ND, Hawkey CJ, Brailsford W, Naesdal J. Gastroduodenal toxicity of low-dose acetylsalicylic acid: a comparison with non-steroidal anti-inflammatory drugs. Curr Med Res Opin. 2009;25(11):2785\u0026ndash;93.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOck CY, Hong KS, Choi KS, Chung MH, Kim Y, Kim JH, et al. A novel approach for stress-induced gastritis based on paradoxical anti-oxidative and anti-inflammatory action of exogenous 8-hydroxydeoxyguanosine. Biochem Pharmacol. 2011;81(1):111\u0026ndash;22.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu B, Cui X, Xu K, Lin L, Zhou Y. Effect of water immersion restraint stress on gastric mucosa in rats with removed salivary glands. Int J Clin Exp Pathol. 2020;13(11):2853\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFan F, Yang M, Geng X, Ma X, Sun H. Effects of Restraint Water-Immersion Stress-Induced Gastric Mucosal Damage on Astrocytes and Neurons in the Nucleus Raphe Magnus of Rats via the ERK1/2 Signaling Pathway. Neurochem Res. 2019;44(8):1841\u0026ndash;50.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYisireyili M, Alimujiang A, Aili A, Li Y, Yisireyili S, Abudureyimu K. Chronic Restraint Stress Induces Gastric Mucosal Inflammation with Enhanced Oxidative Stress in a Murine Model. Psychol Res Behav Manag. 2020;13:383\u0026ndash;93.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrzozowski T, Konturek PC, Mierzwa M, Drozdowicz D, Bielanski W, Kwiecien S, et al. Effect of probiotics and triple eradication therapy on the cyclooxygenase (COX)-2 expression, apoptosis, and functional gastric mucosal impairment in Helicobacter pylori-infected Mongolian gerbils. Helicobacter. 2006;11(1):10\u0026ndash;20.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFu S, Ramanujam KS, Wong A, Fantry GT, Drachenberg CB, James SP, et al. Increased expression and cellular localization of inducible nitric oxide synthase and cyclooxygenase 2 in Helicobacter pylori gastritis. Gastroenterology. 1999;116(6):1319\u0026ndash;29.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Y, Guan WX, Zhou Y, Zhang XY, Zhao HJ. Red ginseng polysaccharide promotes ferroptosis in gastric cancer cells by inhibiting PI3K/Akt pathway through down-regulation of AQP3. Cancer Biol Ther. 2024;25(1):2284849.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim JK, Shin KK, Kim H, Hong YH, Choi W, Kwak YS, et al. Korean Red Ginseng exerts anti-inflammatory and autophagy-promoting activities in aged mice. J Ginseng Res. 2021;45(6):717\u0026ndash;25.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKang H, Lim JW, Kim H. Inhibitory effect of Korean Red Ginseng extract on DNA damage response and apoptosis in Helicobacter pylori-infected gastric epithelial cells. J Ginseng Res. 2020;44(1):79\u0026ndash;85.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBae M, Jang S, Lim JW, Kang J, Bak EJ, Cha JH, et al. Protective effect of Korean Red Ginseng extract against Helicobacter pylori-induced gastric inflammation in Mongolian gerbils. J Ginseng Res. 2014;38(1):8\u0026ndash;15.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim HS, Lim JW, Kim H. Korean Red Ginseng Extract Inhibits IL-8 Expression via Nrf2 Activation in Helicobacter pylori-Infected Gastric Epithelial Cells. Nutrients. 2022;14(5).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNikkhah Bodagh M, Maleki I, Hekmatdoost A. Ginger in gastrointestinal disorders: A systematic review of clinical trials. Food Sci Nutr. 2019;7(1):96\u0026ndash;108.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHaniadka R, Saldanha E, Sunita V, Palatty PL, Fayad R, Baliga MS. A review of the gastroprotective effects of ginger (Zingiber officinale Roscoe). Food Funct. 2013;4(6):845\u0026ndash;55.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbidi C, Rtibi K, Boutahiri S, Tounsi H, Abdellaoui A, Wahabi S, et al. Dose-dependent Action of Zingiber officinale on Colonic Dysmotility and Ex Vivo Spontaneous Intestinal Contraction Modulation. Dose Response. 2022;20(3):15593258221127556.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGaus K, Huang Y, Israel DA, Pendland SL, Adeniyi BA, Mahady GB. Standardized ginger (Zingiber officinale) extract reduces bacterial load and suppresses acute and chronic inflammation in Mongolian gerbils infected with cagAHelicobacter pylori. Pharm Biol. 2009;47(1):92\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSong MY, Lee DY, Park SY, Seo SA, Hwang JS, Heo SH, et al. Steamed Ginger Extract Exerts Anti-inflammatory Effects in Helicobacter pylori-infected Gastric Epithelial Cells through Inhibition of NF-κB. J Cancer Prev. 2021;26(4):289\u0026ndash;97.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSong MY, Lee DY, Park SY, Seo SA, Hwang JS, Heo SH, et al. Erratum: Steamed Ginger Extract Exerts Anti-inflammatory Effects in Helicobacter pylori-infected Gastric Epithelial Cells through Inhibition of NF-κB. J Cancer Prev. 2022;27(1):77.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWagner H. Synergy research: approaching a new generation of phytopharmaceuticals. Fitoterapia. 2011;82(1):34\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWilliamson EM. Synergy and other interactions in phytomedicines. Phytomedicine. 2001;8(5):401\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang W, Li Q, Yan X, Chen Z, Xie Y, Hu H, et al. Comparative study of raw and processed Vladimiriae Radix on pharmacokinetic and anti-acute gastritis effect through anti-oxidation and anti-inflammation. Phytomedicine. 2020;70:153224.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRen WK, Xu YF, Wei WH, Huang P, Lian DW, Fu LJ, et al. Effect of patchouli alcohol on Helicobacter pylori-induced neutrophil recruitment and activation. Int Immunopharmacol. 2019;68:7\u0026ndash;16.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSalim AS. Use of scavenging oxygen-derived free radicals to protect the rat against aspirin- and ethanol-induced erosive gastritis. J Pharm Sci. 1992;81(9):943\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAl-Quraishy S, Othman MS, Dkhil MA, Abdel Moneim AE. Olive (Olea europaea) leaf methanolic extract prevents HCl/ethanol-induced gastritis in rats by attenuating inflammation and augmenting antioxidant enzyme activities. Biomed Pharmacother. 2017;91:338\u0026ndash;49.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHong JB, Zuo W, Wang AJ, Lu NH. Helicobacter pylori Infection Synergistic with IL-1β Gene Polymorphisms Potentially Contributes to the Carcinogenesis of Gastric Cancer. Int J Med Sci. 2016;13(4):298\u0026ndash;303.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLaine L, Jensen DM. Management of patients with ulcer bleeding. Am J Gastroenterol. 2012;107(3):345\u0026ndash;60. quiz 61.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBarrachina MD, Pan\u0026eacute;s J, Esplugues JV. Role of nitric oxide in gastrointestinal inflammatory and ulcerative diseases: perspective for drugs development. Curr Pharm Des. 2001;7(1):31\u0026ndash;48.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDing H, Gao Y, Wang Y, Yao K, Wang G, Zhang J. The role of peripheral serotonin and norepinephrine in the gastroprotective effect against stress of duloxetine. Eur J Pharmacol. 2023;941:175499.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu X, Yuan Z, Luo L, Wang T, Zhao F, Zhang J, et al. Protective role of fruits of Rosa odorata var. gigantea against WIRS-induced gastric mucosal injury in rats by modulating pathway related to inflammation, oxidative stress and apoptosis. Chin Herb Med. 2024;16(2):263\u0026ndash;73.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"Tables are available in the Supplementary Files section."}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-complementary-medicine-and-therapies","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcam","sideBox":"Learn more about [BMC Complementary Medicine and Therapies](https://bmccomplementmedtherapies.biomedcentral.com/)","snPcode":"","submissionUrl":"","title":"BMC Complementary Medicine and Therapies","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Ginseng, Ginger, Traditional food formulation, Stress-induced gastritis, Oxidative stress","lastPublishedDoi":"10.21203/rs.3.rs-8630439/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8630439/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study investigated the gastroprotective effects of a traditional ginseng\u0026ndash;ginger formulation using a water immersion and restraint stress (WIRS)-induced gastritis mouse model. Among the tested mixing ratios, the 3:1 (ginseng:ginger) formulation was the most effective in reducing gastric hemorrhagic lesions. Oral administration of the selected formulation for seven days significantly attenuated stress-induced increases in inflammatory markers, including interleukin-1β, inducible nitric oxide synthase, and cyclooxygenase-2. In addition, the formulation reduced lipid peroxidation while enhancing glutathione peroxidase activity, indicating improved antioxidant defense capacity. Notably, these protective effects were independent of changes in gene expression related to gastric acid secretion. Taken together, the findings suggest that a traditional ginseng\u0026ndash;ginger formulation may contribute to maintaining gastric health under stress conditions through modulation of inflammation- and oxidative stress\u0026ndash;associated responses, supporting its potential relevance as a dietary strategy for maintaining gastric health under stress conditions.\u003c/p\u003e","manuscriptTitle":"Gastroprotective effects of a traditional ginseng–ginger formulation in a stress-induced gastritis mouse model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-28 21:32:37","doi":"10.21203/rs.3.rs-8630439/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewersInvited","content":"","date":"2026-04-20T06:09:44+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-14T20:51:07+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-04-14T20:25:45+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-08T03:25:02+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Complementary Medicine and Therapies","date":"2026-04-08T03:17:05+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-complementary-medicine-and-therapies","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcam","sideBox":"Learn more about [BMC Complementary Medicine and Therapies](https://bmccomplementmedtherapies.biomedcentral.com/)","snPcode":"","submissionUrl":"","title":"BMC Complementary Medicine and Therapies","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"25a0da86-74a3-4521-9282-e2463a2ca208","owner":[],"postedDate":"April 28th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-28T21:32:38+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-28 21:32:37","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8630439","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8630439","identity":"rs-8630439","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}