Efficiency and Mechanism Evaluation of Magnolia officinalis Water Extract in Preventing Gastric Ulcer

In this study we aimed at demonstrating the ability of Magnolia officinalis water extract to ameliorate gastric ulcers in in vitro and in vivo experiments. The gastric mucosa epithelial cell line, RGM 1, was pretreated with Magnolia officinalis water extract (0, 0.1, 1, 2, 5, or 10 mg/ml) and cultured in DMEM/F12 medium (pH 7.4) for 2 h and then in DMEM/F12 medium (pH 4.0) for 10 min. Magnolia officinalis water extract protected the cell viability and decreased reactive oxygen species formation by the acidic medium. In the in vivo experiment, Magnolia officinalis water extract (100 mg/kg) was administrated daily for 28 days in ICR mice via oral gavage, and then Shay's ulcer surgical method was performed to induce gastric ulcers. We analyzed the pH value of stomach acid and the pathological section, inflammation, and cannabinoid receptor type 2 (CB2) cDNA levels of the stomach. Magnolia officinalis water extract not only enhanced the pH value of stomach acid but also ameliorated the ulcer index and inflammation and increased CB2 expression effectively. These results suggest that Magnolia officinalis water extract might be used to decrease the incidence of gastric ulcer.


Introduction
Peptic ulcer disease (PUD) is attributable to multiple etiologies, including genetic characteristics, stress, lifestyle habits, smoking, drug use, and Helicobacter pylori (H. pylori) infection, which have been identifed as major risk factors [1,2]. PUD, consisting of duodenal ulcers and gastric ulcers, is a common disease in the outpatient department of gastroenterology and widely distributed across various ethnic and age groups. Te prevalence of PUD is increased signifcantly in adults who smoke and have a history of longterm medication use [3]. In Taiwan, the prevalence rate of PUD is approximately 10% among people of any age. Moreover, 67% of patients with PUD have no particularly unremarkable symptoms [4]. Finally, proton pump inhibitors (PPIs) and anti H. pylori treatment do not significantly decrease the incidence of PUD [5].
Antacid agents, mucosal protectants (such as sucralfate), H2-antagonists (such as ranitidine), PPIs, and antibiotics of H. pylori are the most common drugs to treat gastric ulcers in modern medicine. However, in Taiwan, people use traditional herbal medicine to treat gastric disorders, and the efcacy of these treatments has been confrmed. For example, Magnolia ofcinalis, also called houpu, is a traditional herbal medicine in Asia for treating gastrointestinal disorders, such as indigestion, fatulence, duodenal ulcers, and gastric ulcers. Several reports have demonstrated that Magnolia ofcinalis ethanol or hexane extract possess several phenolic compounds (such as magnolol, honokiol) that can reduce the production of acidic gastric juice and inhibit the proton pump of gastric parietal cells. Tey also have antioxidation and anti-infammation efects in an ethanol-induced gastric ulcer animal model [6,7].
According to previous studies [6,7], Magnolia ofcinalis extracts were most commonly made with ethanol or hexane. In this study, we investigated the protective efects of a unique and patented Magnolia ofcinalis water extract on gastric mucosa epithelial cells in acidic medium and gastric ulcers induced by Shay's ulcer surgical method in ICR mice.

Magnolia ofcinalis Extraction.
We soaked 100 g of Cortex Magnolia ofcinalis ( Figure 1) in 1000 ml of distilled pure water and boiled it for one hour. After fltering, the solution was evaporated at 60°C water bath in vacuo to remove the solvent (BUCHI Rotavapor R-200, Switzerland) and then lyophilized (EYELA, Freeze Dryer FDU-2100, Japan). Te crystals were stored at −20°C until analysis. Te stock solution formulated 1 g of crystals in 1 ml PBS.

RGM 1 Cells Cultured in Acidic
Medium. Te RGM 1 cell line (RCB0876) was obtained from Prof. Hirofumi Matsui (University of Tsukuba, Japan). RGM 1 cells were cultured in DMEM/F12 medium (pH 7.4) at 37°C in 5% humidity in a CO 2 incubator, and the medium was changed every two days. First, RGM 1 cells were seeded in 96-well plates at a density of 2 × 10 4 cells/well and pretreated with Magnolia ofcinalis water extract (0, 0.1, 1, 2, 5, or 10 mg/ml) or sucralfate (3 mg/ml) as a positive control for 2 h. Te medium was changed to DMEM/F12 medium (pH 4.0) for 40 min to simulate acid damage.

Protection from Magnoliae ofcinalis Water Extract in RGM 1 Cells Cultured in Acidic Medium.
After 40 min of acidic medium stimulation, the cells were incubated for 2 h with 0.5 mg/ml of MTT and dissolved in serum-free medium. Ten, DMSO (100 μl) was added with gentle shaking for 10 min so that complete dissolution was achieved. Aliquots of the resulting solutions were transferred to 96-well plates, and absorbance was recorded at 595 nm using the microplate spectrophotometer system (Spectra max190-Molecular Devices). Te results were analyzed with the Soft max pro software (version 2.2.1) and are presented as percentages of the control values.

ROS Production of RGM 1 Cells Cultured in Acidic
Medium. We used 0.1, 1, and 2 mg/ml of Magnolia ofcinalis water extract to perform the experiment. RGM 1 cells were seeded in 24-well plates at a density of 2 × 10 5 cells/well and pretreated with Magnolia ofcinalis water extract (0, 0.1, 1, or 2 mg/ml) for 2 h. Ten, the medium was changed to DMEM/F12 medium (pH 4.0) for 10 min. We then washed with PBS and added 10 μM 2,7-dichlorofuorescein diacetate (H2DCF-DA, Invitrogen, USA) dissolved in PBS. Ten, the cells were incubated at 37°C for 30 min. After removing the solution, the cells were incubated in MEM medium (without phenol red) for 5 min and the cells were collected. Te positive cell counts and mean fuorescence intensities were analyzed by fow cytometry (Beckman Coulter Epics XL-MCL, USA) using 488 nm excitation and 526 nm receiving fuorescence signals.

Animal Model of Gastric Ulcer.
Tis study was approved by the Animal Care and Use Committee of National Pingtung University of Science and Technology (approval number: IACUC-NPUST-106-053). Shay's rat ulcer model was adopted and modifed [8]. Normal six-week-old ICR mice were purchased from BioLASCO Taiwan Co., Ltd. (Taipei, Taiwan) and housed under constant temperature, humidity, and illumination (12 hours of light and dark). Water and standard diet were made available ad libitum. After an adaptation period, the mice were divided into four groups (n � 10, each group). One group was a sham group, and the other groups were administrated daily with saline, Magnolia ofcinalis water extract (100 mg/kg), or ranitidine (100 mg/kg) via oral gavage for 28 days.
Te mice were fasted on day 28. After the pretreatment period of 1 h on the last day, the mice were anesthetized with Zoletil (50 mg/kg) (Virbac, New Zealand). Te abdomen was opened by a small midline incision below the xiphoid process, and the pylorus portion of the stomach was slightly lifted out and ligated by surgical sutures. Precaution was taken to avoid traction to the pylorus or damage to its blood supply. Te stomach was placed carefully in the abdomen and the wound was sutured by interrupted sutures. Ten, 4 h after pylorus ligation, the mice were euthanized by cervical dislocation after anesthetization, and the stomach was removed. Te volume, pH, and total acidity of gastric fuid were determined. Te stomach was then incised along the greater curvature and observed to elucidate the ulcer index. Te ulcer index was counted using a magnifying glass, and the diameter of the ulcers was measured using a vernier caliper.

Ulcer Index Determination.
Te ulcer index was modifed by following the scoring method of Suzuki et al. [9] and judged by Prof. Ching-Dong Chang (pathologist, National Pingtung University of Science and Technology). Te formula was as follows: Protection ration (%) � (Control ulcer index -Test ulcer index)/Control ulcer index.

Statistical
Analysis. All data were analyzed to determine the statistical signifcance of diferences among groups. Student's t-test, Mann-Whitney U test, and one-way analysis of variance with Bonferroni's post hoc test were used to assess the diferences between groups. P < 0.05 was considered statistically signifcant. Te Statistical Package for Social Sciences (SPSS, version 19.0; Chicago, IL, USA) was used to perform all statistical analyses.
When RGM 1 cells were cultured in acidic medium for 10 min, the cell viability was signifcantly decreased. After pretreatment with sucralfate (3 mg/ml) or Magnolia ofcinalis water extract (0.1, 1, or 2 mg/ml), the cell viability of RGM 1 cells was increased in the acidic medium stimulation, while 5 and 10 mg/ml of Magnolia ofcinalis water extract did not increase the cell viability ( Figure 2). Terefore, we suggest that Magnolia ofcinalis water extract in low doses, as well as sucralfate, which possessed protective efects for gastric mucosa epithelial cells damaged by gastric acid.

Antioxidant Efect of Magnolia ofcinalis Water Extract In
Vitro. Te reactive oxygen species (ROS) production of RGM 1 cells induced by acidic medium for 10 min was more signifcant than that for 40 min (Table 1). We found that 0.1 mg/ml of Magnolia ofcinalis water extract and 3 mg/ml of sucralfate efectively decreased the ROS production (Table 2). However, Magnolia ofcinalis water extract did not exhibit dose-dependent efcacy in this test. Figure 3 shows the modifed scoring method of the ulcer index, which could more easily assesss the severity of gastric ulcers. After pylorus ligation for 4 h, the gastric ulcers of the saline group were induced signifcantly, compared with those of the sham group. However, in the ranitidine or Magnolia ofcinalis water extract group, the gastric ulcers were reduced signifcantly. Terefore, Magnolia ofcinalis water extract as well as ranitidine, ameliorated gastric mucosa damage from gastric acid (Table 3).

Biochemical Parameters In Vivo.
After pylorus ligation for 4 h, the gastric juice secretion and pH value of gastric juice in the saline group were higher than those of the sham group (Table 3). Terefore, the pylorus ligation was successful for inducing gastric ulcers. In contrast, in the ranitidine or Magnolia ofcinalis water extract groups, those parameters were signifcantly ameliorated (Table 3). CB2 activation is known to reduce infammation. Although the CB2 gene expression was not changed by pylorus ligation between the saline and sham groups, ranitidine or Magnolia ofcinalis water extract could enhance CB2 gene expression (Table 4). Meanwhile, after pylorus ligation, the concentrations of the infammatory cytokines, TNF-α and IL-6 were increased. Ranitidine or Magnolia ofcinalis water extract reduced the levels of those cytokines signifcantly (Table 4). Terefore, ranitidine or Magnolia ofcinalis water extract might regulate the infammation through stimulating CB2 gene (Table 4).

Discussion
Tis study demonstrated that Magnolia ofcinalis water extract could prevent the RGM 1 gastric mucosa epithelial cell line from being damaged by gastric acid. It could also ameliorate gastric acid-induced gastric ulcers and infammation in mice. Tese fndings prove the therapeutic benefts of Magnolia ofcinalis water extract in treating gastric ulcers.
Magnolia ofcinalis is applied widely in Asian traditional medicine for its multiple uses, including sedative, antispastic, antibiotic, antioxidant, and antiinfammatory efects, as well as for its ability to treat tract ulcer diseases [10,11]. Te major substances of Magnolia ofcinalis, magnolol and honokiol, are responsible for its benefcial properties. Many food safety authorities have evaluated Magnolia ofcinalis and its extract and indicated their safety. Furthermore, Sarrica et al. [11] demonstrated that using Magnolia ofcinalis and its extract for more than one year did not result in considerable adverse efects. Additionally, our result demonstrated that Magnolia ofcinalis water extract (a) (b) Figure 1: An introduction to Magnolia ofcinalis (houpu). Magnolia ofcinalis, also called houpu, is a traditional herbal medicine in Asia for treating gastrointestinal disorders, like indigestion, fatulence, duodenal ulcer, and gastric ulcer. Firstly, to collect the tree bark and root bark from Magnolia ofcinalis and to dry it (a), after soaking with ginger juice for a period of time, that is a complete medicinal material for clinical use (b). In this study, the houpu we used was soaked with ginger juice for a period of time.
(0.1-10 mg/ml) had no cytotoxicity in the RGM 1 cell line, which was consistent with the fndings of Sarrica et al.
Under the stimulation of excessive gastric acid secretion for a prolonged period, the gastric mucosa will experience serious damage, eventually resulting in gastric ulcers and perforation [12]. Protecting the gastric mucosa is benefcial for treating ulcers. Sucralfate, a mucosal protectant, is an aluminum salt of sucrose octasulfate that can be activated with gastric acid to form a viscous material that acts as an acid bufer. Tis protectant attaches to proteins on the surface of the ulcers to form a stable complex to prevent further gastrointestinal ulcers from gastric acid, pepsin, or bile acid [13]. Sucralfate can also stimulate the production of prostaglandins and gastric mucus to protect the tract [14]. In this study, Magnolia ofcinalis water extract, as well as sucralfate, could protect the RGM 1 cell line from damage induced by an acidic medium. Terefore, we suggest that Magnolia ofcinalis water extract could be used as a mucosal protectant like sucralfate.
Te major risk factors of peptic ulcers, such as stress, smoking, alcohol, and drugs are related to tissue damage caused by ROS production [15]. When RGM 1 cells were cultured in acidic medium, ROS production was observed. However, after treating these cells with Magnolia ofcinalis water extract, ROS generation was inhibited. We also confrmed that sucralfate could decrease ROS production, which was consistent with the fndings of Sato et al. [16]. We believe that one of the mucosal protective mechanisms of Magnolia ofcinalis water extract is related to inhibiting ROS generation.
In this study, we performed pylorus ligation surgery to induce gastric ulcers and observed the results of Shays' method. After pretreatment with Magnolia ofcinalis water extract or ranitidine, the ulcer index was decreased signifcantly, indicating that both of these treatments could inhibit gastric acid-induced gastric ulcers. Furthermore, in the analysis of the tissue biochemical parameters, the CB2 gene expression was not changed signifcantly, but TNF-α and IL-6 concentrations were increased by pylorus ligation surgery. Cannabinoid receptor (CB) can be roughly divided into two types: CB1 and CB2. CB1 receptor is predominantly expressed in the central nervous system (CNS) to mediate the   psychotropic and behavioral efects of cannabinoid. In comparison, CB2 receptor is widely distributed in several tissues, such as CNS, cardiovascular, respiratory, hepatic, and reproductive systems to afect their function and immunity. In the gastrointestinal tract, CB2 modulates visceral motility, sensitivity, and infammation. Te downregulation of CB2 is proved to enhance gastric infammation and ulcers [17]. Some reports have demonstrated that Magnolia ofcinalis can activate CB2 [18,19]. In this study, we were the frst to prove that Magnolia ofcinalis water extract and ranitidine could stimulate CB2 gene expression and inhibit infammatory   cytokines in a mouse model of gastric ulcers. We believe this to be direct evidence of the mechanism of Magnolia ofcinalis in treating gastrointestinal ulcers.

Conclusion
Magnolia ofcinalis water extract exhibited gastric mucosal protection, inhibited ROS production, prevented ulcer formation, and reduced infammation in gastric ulcers ( Figure 4). We developed the water extraction of Magnolia ofcinalis and proved its efciency and mechanisms in preventing gastric ulcers. We believe our fndings will contribute to a wider application of Magnolia ofcinalis in traditional medicine.

Data Availability
Te data used to support the fndings of this study are available from the corresponding author upon reasonable request.

Ethical Approval
Tis study was approved by the Animal Care and Use Committee of National Pingtung University of Science and Technology (approval number: IACUC-NPUST-106-053).

Conflicts of Interest
Te authors declare to have no confict of interest regarding this article. water extract not only enhanced the pH value of stomach acid but also ameliorated the ulcer index and infammation and increased CB2 expression efectively. Tese results suggest that Magnolia ofcinalis water extract might be used to decrease the incidence of gastric ulcer. 6 Evidence-Based Complementary and Alternative Medicine