Introduction

Gastric ulcers are a common type of gastrointestinal disease and exhibit a high prevalence rate. Studies have shown that the etiology of ulcers is associated with cigarette smoking, stress, infections, nutritional deficiencies, frequent ingestion of non-steroidal anti-inflammatory drugs, and alcohol consumption (Arab et al. 2015; Sidahmed et al. 2019). Among these factors, excessive alcohol consumption has attracted the most attention, as it has been demonstrated to considerably increase the incidence of gastric ulcers (Yamada et al. 2005). However, the pathogenic mechanisms underlying ethanol-induced gastric ulcers have not been fully clarified.

Previous studies have shown that ethanol is a known cause of gastric damage by reducing mucosal protein secretion and prostaglandin E2 (PGE2) production (Sidahmed et al. 2015; Sidahmed et al. 2019), as well as by increasing disruptive factors such as oxidative stress and pro-inflammatory cytokines (Li et al. 2018; Sidahmed et al. 2019). Alcohol disrupts the expression of cytokines, including tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), in the pathogenesis of gastric ulcers (Chen et al. 2016; Liu et al. 2016). Thus, controlling the formation of reactive oxygen species (ROS) and inflammatory reactions are essential for the treatment of this pathology. Another key defensive factor against gastric ulcers is the production of gastric mucus. A sufficient amount of gastric mucus can potentiate the action of a protective agent on the gastric mucosa. Therefore, it is an effective protection and a method of treatment to directly or indirectly balance the damage and defense factors of gastric ulcers.

Current treatments for gastric ulcers mainly include the administration of proton-pump inhibitors, H2-receptor antagonists, gastric mucosa protective agents, and antibacterial drugs that kill Helicobacter pylori (Kuna et al. 2019). However, these treatments are ultimately not effective in preventing ethanol-induced gastric ulcers and have considerable severe side effects during long-term use (Kuna et al. 2019). Thus, there is an urgent need to identify and develop more effective treatments for ameliorating gastric ulcers.

Schisandra chinensis (Turcz.) Baill., Schisandraceae, also known as Chinese magnolia vine (“wuweizi,” or five-flavor-fruit), is a medicinal plant that has been extensively used in traditional and modern medicine in China, Japan, and Russia (Hernandez et al. 1988; Panossian and Wikman 2008; Szopa et al. 2017). The fruit of S. chinensis (magnolia berry) is used as a tonic, sedative, antitussive, and anti-aging remedy (Dilshara et al. 2013). The components of S. chinensis that have exhibited pharmacological activities are lignans with a dibenzocyclooctadiene skeleton, such as schisandrin (1), schisantherin A (2), deoxyschisandrin (3), and γ-schisandrin (4). These pharmacological activities included antioxidative, anti-hepatotoxic, anti-ulcerogenic, and anti-inflammatory properties (Hernandez et al. 1988; Huyke et al. 2007; Kang et al. 2014; Szopa et al. 2017). Further studies have shown that the components of magnolia berries exhibit inhibitory effects on the expression levels of TNF-α and IL-1β by deactivation of the mitogen-activated protein kinase pathway (Giridharan et al. 2015), which subsequently prevents the production of pro-inflammatory mediators and cytokines. Furthermore, several previous studies have reported efficacies of this medicinal plant in mitigating central nervous system disorders (e.g., depression, neurosis, and alcoholism), hepatotoxic disease, and peptic ulcers (Panossian and Wikman 2008; Szopa et al. 2017). In addition, other studies have shown that S. chinensis extracts, particularly through component lignans or formulations containing deoxyschizandrin, exhibit inhibitory effects on stress-induced gastric ulcers (Hernandez et al. 1988). However, the protective effects and mechanisms of S. chinensis extracts on ethanol-induced gastric ulcers have remained unclear, although such extracts have been shown to be effective in the treatment of inflammation and oxidative stress in many other diseases.

In animal experiments, an ethanol-induced gastric ulcer model is often used to screen for compounds that possess anti-ulcer activities (Yang et al. 2017; Li et al. 2018; Sidahmed et al. 2019). Therefore, the aim of the present study was to investigate the effects and possible use of magnolia berry alcohol extracts from S. chinensis against ethanol-induced gastric ulcers in mice by measuring the activities of antioxidative substances, levels of cytokines, and pathological changes in gastric mucosa.

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Materials and Methods

Preparation of Extracts

Berries of Schisandra chinensis (Turcz.) Baill., Schisandraceae, were purchased from Zhejiang Zhenyuan Pharmaceutical Co., Ltd. (Shaoxing, China) and identified by Professor Xiuzhen Wang, Faculty of Medicine and Health, Yuanpei College, Shaoxing University, as the dried and mature fruit. In this experiment, the botanical material was stored in a dry, dark, and low-temperature place and a sample was kept in our laboratory for future reference. The purchased samples were dried and homogenized to a fine powder using a grinder before extraction. The powder (30 g) was placed into a round bottom flask and was reflux extracted with 80% ethanol at a material-to-liquid ratio of 1 g per 10 ml at 90 °C for 1 h. This procedure was repeated three times, and the resultant solution was then filtered and concentrated using a rotary vacuum evaporator. The solution was dried in a drying cabinet, resulting in a crude extract (7.29 g) that was stored at 4 °C and diluted with medium to the desired concentration before use.

Preparation of Solutions

For the preparation of the corresponding solutions for each reference compound, 10.3 mg of schisandrin (1), 4 mg of schisantherin A (2), 6.5 mg of deoxyschisandrin (3), and 10.4 mg of γ-schisandrin (4) were individually dissolved in 25 ml of methanol. Additionally, 559.5 mg of crude extract was dissolved in 100 ml of methanol.

Quantification of Chemical Marker

In the present study, we used an Agilent 1200 HPLC instrument. Chromatographic conditions were as follows: C18 column, 4.6 × 250 mm, 5 μm; mobile phase, A, H2O; mobile phase B, MeOH; gradient elution was with 60–100% mobile phase B from 0 to 15 min, 100% mobile phase B from 15 to 20 min, and 100–60% mobile phase B from 20 to 25 min; flow rate was 0.5 ml/min and the oven temperature was set at 45 °C; and detection was at 280 nm and the injection volumes of standards and extracts were 5 μl each (He et al. 1997; Lee and Kim 2010). Aqueous methanolic stock solutions containing the four analytes were prepared by mixing compounds 1–4 in a 1:1:1:2 volumetric ratio and diluting them to the appropriate concentrations (5%, 10%, 20%, 50%, 80%, and 100%) for the establishment of calibration curves. Six different concentrations of the mixed analyte solutions were injected in triplicate. Variations were expressed as relative standard deviations (RSDs).

Induction of Gastric Ulcers and Treatments

After 1-week adaptation to the environment, mice were randomly divided into 6 groups with 10 mice in each group. In group 1 (control), mice received vehicle (0.9% physical saline). In group 2 (ethanol), mice received absolute ethanol (10 ml/kg) (Li et al. 2018). In group 3 (omeprazole), mice received omeprazole before the induction of ulcers at a dose of 20 mg/kg. Finally, mice in groups 4–6 (SC80–320) received the analyzed extracts before the induction of ulcers at doses of 80, 160, and 320 mg/kg for groups 4, 5, and 6, respectively. Mice were fasted for 24 h before the experiment, during which they were provided free access to drinking water. The induction of ulcers was achieved by oral administration (p.o.) of absolute ethanol at a dose of 10 ml/kg body weight, while the control group received normal saline instead. One hour later, blood was collected by removal of the eyes. Then, the collected blood was centrifuged for 10 min at 2110×g to obtain clean plasma that was stored at − 20 °C until further use. Finally, mice were sacrificed by dislocating their necks. A diagram of the study design is shown in Fig. S1 (Supplementary Material).

Determination of Gastric Ulcer Index

After the mice were euthanized, their stomachs were removed and examined for the observation of mucosal hemorrhagic lesions macroscopically. Each lesion’s length, in millimeters, was carefully measured to determine the mean ulcer index (UI). Mucosal lesions were evaluated as follows: no ulcer (0), 1–5 petechiae (< 1 mm) (1), 6–10 petechiae (< 1 mm) (2), > 10 petechiae (< 1 mm) (3), small ulcer (< 2 mm) (2), medium ulcer (2–4 mm) (3), and large ulcer (> 4 mm) (4). If the width was > 1 mm, the points were multiplied by a factor of 2. The UI was equal to the sum of all scores dividing by the number of mice (Li et al. 2018).

Determination of Plasma Malondialdehyde Levels and Superoxide Dismutase Activities

The plasma was centrifuged at 2110×g for 10 min at 4 °C, after which plasma malondialdehyde (MDA) levels and superoxide dismutase (SOD) activities were assayed by immunoassay kits according the manufacturer’s instructions (Mohan et al. 2020).

Immunoassay Analysis

Plasma TNF-α and IL-1β levels were assayed using monoclonal antibodies for TNF-α and IL-1β, in accordance with the instructions of ELISA kits (Li et al. 2018).

Statistical Analysis

All values are expressed as the mean ± standard error of the mean (SEM). The SPSS statistical software (SPSS 18.0) was used for all statistical analysis. The data were statistically analyzed by one-way analyses of variance (ANOVAs) followed by Newman-Keuls tests or Dunnett’s multiple comparison tests for post hoc pairwise comparisons. A p < 0.05 was considered statistically significant.

Results

HPLC Analysis

Four reference compounds, schisandrin (1), schisantherin A (2), deoxyschisandrin (3), and γ-schisandrin (4), were subjected to HPLC analysis to determine their retention times. Figure 1 shows the HLPC-UV chromatograms of mixed standards and the crude extracts from magnolia vine berries; the results showed that the total extract contained the four chemical markers used to obtain the chemical profile. To determine the concentrations of these main components, HPLC was used to establish a standard curve, verify linear ranges, and compute correlation coefficients (R2) (Table 1). The concentrations of compounds 1–4 were 3.57 ± 0.353, 0.94 ± 0.123, 0.63 ± 0.017, and 2.61 ± 0.064 mg/g, respectively; these results are consistent with previously reported findings (Lee and Kim 2010).

Fig. 1
figure 1

HPLC-UV chromatograms of mixed chemical markers used as standards (a) and crude extracts from berries of Schisandra chinensis (b). Reference compounds: 1 schisandrin; 2 schisantherin A; 3 deoxyschisandrin; and 4 γ-schisandrin

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Table 1 Linear regression data and precision of investigated compounds from Schisandra chinensis extracts
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Evaluation of Gastric Lesions

Gross examinations of the stomachs of mice among the groups are illustrated in Fig. S2. As shown in Fig. S2A, no macroscopic or microscopic lesions were observed in the control group. Compared with the features of the control group, mice treated with absolute ethanol (10 ml/kg) induced macroscopic morphological changes, such as linear hemorrhagic lesions and multifocal mucosal erythema (Fig. S2B). Compared with features in the ethanol-induced ulcer group, mice pretreated with omeprazole (Fig. S2C) and crude extract (Fig. S2D–F) had significantly diminished areas of gastric damage formation and suppression of the formation of hemorrhagic mucosal lesions. The most prominent protection of the gastric mucosa was observed at the middle dose of 160 mg/kg of the crude extract (Fig. S2E). Compared with that of the vehicle group, the group treated with absolute ethanol (10 ml/kg) displayed a marked increase in the UI (p < 0.001) (Fig. 2). Pretreatment with omeprazole (20 mg/kg, i.g.) or the tested extract (80, 160, or 320 mg/kg, i.g.) significantly decreased the UI in the ethanol-induced gastric ulcer groups in comparison with that of the ethanol group. Lesions in the gastric mucosa were significantly reduced in mice pretreated with the extract of S. chinensis at doses of 320 mg/kg (4.9 ± 1.069, p < 0.001), 160 mg/kg (7.9 ± 1.418, p < 0.001), and 80 mg/kg (9.7 ± 1.856, p < 0.01) (Fig. S2D–F). Omeprazole (20 mg/kg), used as the reference drug, also significantly reduced gastric lesions (4.9 ± 0.971, p < 0.001) when compared with that of the ulcer control group (16.8 ± 1.323, p < 0.001).

Fig. 2
figure 2

Effects of the crude extract of Schisandra chinensis on the gastric UI in the following groups: the saline-treated group (control); ethanol group (10 mg/kg); omeprazole (20 mg/kg) group; S. chinensis (80 mg/kg) group (SC80); S. chinensis (160 mg/kg) group (SC160); and S. chinensis (320 mg/kg) group (SC320). Each value represents the mean ± SEM from 10 mice. Significance is represented as ***p < 0.001 compared with the control group, and ##p < 0.01 and ###p < 0.001 compared with the ulcer control group

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Mice treated with absolute ethanol exhibit a significant reduction of thickness in mucosa, a damage of the surface epithelium with epithelial cell deficiency, intense edema, and leucocyte infiltration in the submucosal layer (Fig. 3). As the positive control, omeprazole exhibited relative protection against the effects of ethanol, as observed by an improvement in the superficial region, decreased edema in the submucosa, and only moderate infiltration of neutrophilic inflammatory cells (Fig. 3c). In contrast, pretreatment with S. chinensis (Figs. 3d–f) yielded protection against ethanol-induced histopathological disruption and nearly normal mucosal morphological characteristics.

Fig. 3
figure 3

HE staining of gastric mucosa in mice (× 100). a The control group is shown. b The absolute ethanol group (10 ml/kg) is shown. Broad erosion (gray arrow) in the upper half of mucosa, extensive edema (black arrow) in the submucosal layer, and encircling by the leukocyte inflammatory zone (black arrow) were observed. c The omeprazole group (20 mg/kg) is shown. Mild disruption of the surface epithelial mucosa was present, but deep mucosal damage was absent. d The Schisandra chinensis (80 mg/kg) group is shown. There were no deep ulcers in the gastric mucosa, but there was partially infiltration by inflammatory leukocytes. e, f The S. chinensis (160 and 320 mg/kg) groups are shown, respectively. There were no disruption to the surface epithelium, no edema, and no leukocyte infiltration into the submucosal layer

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Mucosal Defense Mechanisms

Sections of gastric mucosa from control mice revealed periodic acid-Schiff (PAS)–positive reactions (Fig. S3A). In contrast, in the alcohol-treated group, there was a PAS-negative reaction in areas correlated with mucosal damage (Fig. S3B). Both the crude extract and omeprazole treatment groups showed a strong PAS reaction (Figs. S3C–F). These results demonstrated that gastric mucosal glycoprotein in mice treated with absolute ethanol was markedly depleted. However, pretreatment with the crude extract or omeprazole significantly preserved mucosal glycoprotein. At a dose of 320 mg/kg, the analyzed extract showed the greatest intensity in PAS staining (Fig. S3).

Antioxidative and Anti-inflammatory Effects of Drug Administrations

Oral treatment of mice with absolute alcohol (10 ml/kg) significantly increased MDA levels and reduced SOD levels in plasma compared with those in the control group (p < 0.05). Furthermore, in the SC160 and SC320 groups, the MDA levels showed a significant decrease and the SOD levels displayed a partial prevention of depletion in comparison with these parameters in the model group (p < 0.001 and p < 0.05). However, the plasma levels of MDA and SOD in the SC80 group were not significantly different from those in the ethanol-treatment group. Furthermore, omeprazole at 20 mg/kg prevented the ethanol-induced increase in lipid peroxidation (p < 0.05) and decrease in SOD levels (p < 0.05) (Fig. 4).

Fig. 4
figure 4

Effects of the crude extract of Schisandra chinensis on the plasma levels of MDA (a) and SOD (b) in ethanol-induced gastric ulcers in mice. Groups are as defined in Fig. 2. Each value represents the mean ± SEM of 10 mice. Significance is represented as *p < 0.05 and **p < 0.01 compared with the control group, and #p < 0.05 and ##p < 0.01 compared with the ulcer control group

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The administration of ethanol significantly increased the levels of plasma TNF-α and IL-1β by 24% and 32%, respectively (p < 0.001), when compared with those in the control group. As shown in Fig. 5, the plasma levels of TNF-α and IL-1β were increased markedly in the ulcer control group (p < 0.01 and p < 0.05, respectively) when compared with those in the control group. In contrast, pretreatment of ethanol-treated mice with either omeprazole or the crude extract at doses of 80, 160, and 320 mg/kg caused a significant decrease in plasma TNF-α levels by 20%, 33%, 41%, and 50%, respectively (p < 0.01, p < 0.01, p < 0.001, and p < 0.001) and a decrease in IL-1β levels by 27%, 35%, 49%, and 56%, respectively (p < 0.05, p < 0.001, p < 0.001 and p < 0.001) (Fig. 5).

Fig. 5
figure 5

Effects of the crude extract of Schisandra chinensis on the serum levels of IL-1β (a) and TNF-α (b) in the ethanol-induced gastric ulcers in mice. Groups are as defined in Fig. 3. Each value represents the mean ± SEM of 10 mice. Significance is represented as *p < 0.05 and **p < 0.01 compared with the control group, and #p < 0.05, ##p < 0.01, and ###p < 0.001 compared with the ulcer control group

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Discussion

In the present study, we explored the gastroprotective effects of crude total extracts from magnolia vine berries on ethanol-induced gastric lesions in mice. Our results showed that pretreatment with these extracts dramatically ameliorated hemorrhagic superficial gastric lesions and histological signs of damage, as well as decreased pro-inflammatory cytokines (i.e., TNF-α, IL-1β) and plasma MDA levels while increasing plasma SOD activity and gastric wall mucosal glycoprotein production. Collectively, our findings reveal that ethanol extracts from the berries of S. chinensis have potential in mitigating ethanol-induced gastric lesions.

Gastric mucus is the first line of defense against gastric acid, and it prevents gastric mucosa from self-digestion. In the present study, extracts of S. chinensis significantly increased the glycoprotein production of mucus (Fig. S3). Our results were further supported by PAS-staining experiments, which confirmed characteristic carmine staining of stomach regions that secrete mucopolysaccharides (Fig. S3). The tissue sections from mice pretreated with extracts showed an intense secretion of mucus in gastric glands, reflecting a higher secretion in these glands. Consequently, the extracts at a dose of 320 mg/kg contributed to the repair of gastric mucosal damage by improving the gastric wall mucus.

Furthermore, it has been widely reported that oxidative stress has an involvement in the pathogenesis of ethanol-induced gastric ulcers (de Araujo et al. 2018; Sidahmed et al. 2019; Mohan et al. 2020). Specifically, oxidative stress plays a key role in both gastric hemorrhages and the formation of ulcers. SOD is an important antioxidative enzyme that converts hydrogen peroxide to oxygen and water. Therefore, the investigation of antioxidants, such as by assessing SOD activity, may help to elucidate the mechanisms of S. chinensis extracts, which we found also increased the antioxidative activity of SOD enzymes. Studies in isolated organs, tissues, cells, and enzymes have revealed that S. chinensis exhibits strong antioxidative activities. This activity may be attributed to its component ingredient, deoxyschisandrin, which inhibits H2O2-induced apoptosis in intestinal epithelial cells through nuclear factor-kappa B (Gu et al. 2010). In the present study, experimental groups had elevated SOD levels after the administration of the crude extracts. Therefore, our present results demonstrate that S. chinensis may reduce oxidative stress and enhance antioxidative protection.

Another primary factor in ethanol-induced gastric damage is ROS-mediated lipid peroxidation (Sidahmed et al. 2019; Mohan et al. 2020). As mentioned above, the elimination of ROS has been regarded as one of the mechanisms involved in ulcer healing. In addition, the formation of ROS is related to an increase in lipid peroxidation. MDA is the final product of lipid peroxidation and is widely used to determine the levels of peroxidation lipids; hence, the level of MDA represents a biomarker of oxidative stress. For this reason, we investigated the effects of S. chinensis on MDA levels in ethanol-induced ulcerous stomach tissue in mice. We found reduced MDA levels in mice treated with S. chinensis extracts compared with those in ethanol group. Thus, S. chinensis extracts may generate gastroprotective effects by decreasing lipid peroxidation products. In addition to above factors, the gastroprotective effects of S. chinensis extracts may be due to inhibition of inflammatory mediators such as TNF α and IL-1β. Numerous findings have shown that TNF-α and IL-1β levels are dramatically increased in the gastric tissues of ethanol-induced ulcers (Alzokaky et al. 2020; Mohan et al. 2020). These pro-inflammatory cytokines have been reported to play an important role in ethanol-induced gastric ulcer formation, particularly TNF-α, which is a representative inducer of gastric mucosal apoptosis by activating the caspase family of proteases. Much evidence has supported that high-plasma TNF-α levels induce leukocyte adherence, whereas TNF-α antibodies reduce leukocyte adherence and ameliorate gastric mucosal damage (Liu et al. 2016; de Araujo et al. 2018 ). Similarly, IL-1β, another key pro-inflammatory cytokine participating in inflammation, modulates the expression of genes involved in cell cycle progression and inhibition of apoptosis. Our present study demonstrated that administration of S. chinensis extracts inhibited the production of TNF-α and IL-1β in ethanol-induced gastric ulcers and decreased gastric lesions by alleviating gastric mucosal inflammation. In addition, we found that the effects of high-dose S. chinensis extracts (320 mg/kg) on TNF-α and IL-1β levels were similar to those of omeprazole.

Conclusions

In conclusion, our present findings suggest that pretreatment with S. chinensis extracts suppressed ethanol-induced gastric damage in mice by ameliorating inflammation and oxidative stress, as well as by preserving mucous glycoprotein levels in the gastric mucosa. Furthermore, our findings also indicate that S. chinensis may be a promising anti-gastric ulcer drug to protect the stomach from acute tissue lesions induced by ethanol or other harmful factors.