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β-Glucan-enriched fermented barley bran (Sigumjang meju) extracts attenuates gastric mucosal injury induced by acute alcohol intake in vivo
Food Bioscience 28 (2019) 20–26
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β-Glucan-enriched fermented barley bran (Sigumjang meju) extracts attenuates gastric mucosal injury induced by acute alcohol intake in vivo
T
Hojeong Jeonga,1, Dongyeop Kima,b,1, Hyo Jin Songa, Soohyung Leea, Mihyung Kima, ⁎ Keuk-Jun Kimc, Gi Dong Hana, a
Department of Food Science and Technology, Yeungnam University, Gyeongsan, Republic of Korea School of Dental Medicine, University of Pennsylvania, Philadelphia, PA, USA c Department of Clinical Pathology, Daekyeung University, Gyeongsan, Republic of Korea b
A R T I C LE I N FO
A B S T R A C T
Keywords: Fermented barley bran Bacillus spp. β-glucans Gastric mucosal injury Alcohol Sigumjang
Fermented barley bran (FBB) using Bacillus spp. is the main ingredient of sigumjang, a traditional fermented food in South Korea. The aim of this study was to evaluate the gastro-protective effects of β-glucan-enriched FBB extracts in an experimental mouse model of the EtOH-induced gastric ulcer. The gastro-protective activity of FBB was determined using histopathological inspection, level of EtOH absorption from the gastrointestinal-tract and measurement of pro-inflammatory cytokine. Pretreatment of FBB showed less gastrointestinal bleeding and reduced EtOH absorption into the bloodstream with the gastric mucosa. Furthermore, FBB effectively reduced the EtOH-induced mRNA expression of tumor necrosis factor α in the gastric mucosa and its level in the blood. These results showed that FBB can prevent gastric mucosal damage induced by acute EtOH-administration in mice by reducing absorption of EtOH by gastric mucosa along with decreasing the inflammatory response. Altogether, this data provides useful information on how β-glucan-enriched food modulates the EtOH-induced gastric mucosal damage, and thus, it could lead to the development of formulations for protecting gastric mucosal injury with acute alcohol intake.
1. Introduction Alcohol abuse is a devastating illness that has a public impact in the general population (Holden, 1987). Alcohol abuse has many long-term effects that result in premature death, causing 5.9% of all deaths globally. It also increases the propensity for serious illness and accounts for 5.1% of the global burden of alcohol-attributable disease and injury (Rehm et al., 2009; World Health Organization, 2014). The gastrointestinal (GI) tract is the primary digestive organ system for alcohol metabolism and absorption into the bloodstream (Bode & Bode, 1997). The direct contact of alcohol with the mucosa can induce numerous metabolic and functional changes, leading to mucosal damage, such as acute GI bleeding and diarrhea (Bode & Bode, 1997). The consumption of excessive alcohol causes necrotic lesions in the gastric mucosa due to acute hemorrhagic lesions, mucosal edema, epithelial exfoliation, and inflammatory cell infiltration, which in turn, reduces defensive factors such as bicarbonate secretion and mucus production (Choi et al., 2016; Guslandi, 1987). Furthermore, excessive
consumption of alcohol is responsible for a wide range of diseases, including alcoholic hepatitis, cirrhosis and hepato-carcinoma, pancreatitis, cardiomyopathy, hypertension, stroke, and fetal alcohol syndrome (Wang et al., 2016). In alcoholic organ damage, inflammation leads to an increase in the levels of inflammatory cytokines such as tumor necrosis factor α (TNFα), transforming growth factor α (TGF-α), interleukin 6 (IL-6), and interleukin 10 (IL-10). In particular, acute alcohol intake results in high levels of TNF-α in the bloodstream, suggesting that this proinflammatory cytokine has an important role in the pathogenesis of alcohol-induced acute gastric damage (Liu, Liu, & Liu, 2013). Hence, gastric mucosal protecting approaches modulating alcohol absorption, and thereby limiting the risk of the inflammatory response induced using an excessive amount of alcohol could be a potentially effective treatment to minimize the damage of acute alcohol intake. Barley bran is recognized as an alternative or complementary medicine. For example, it has been used for lowering blood sugar, blood pressure and cholesterol (Idehen, Tang, & Sang, 2017). Furthermore, it
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Correspondence to: Department of Food Science and Technology, College of Life and Applied Sciences, Yeungnam University, 280 Daehak-Ro, Gyongsan, Gyeongbuk 58541, Republic of Korea. E-mail address: [email protected] (G.D. Han). 1 These authors contribute equally as co-first author. https://doi.org/10.1016/j.fbio.2019.01.007 Received 10 April 2018; Received in revised form 4 January 2019; Accepted 12 January 2019 Available online 15 January 2019 2212-4292/ © 2019 Elsevier Ltd. All rights reserved.
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2.2.3. Quantification of water-soluble β-glucan Contents of low-molecular weight and water-soluble β-glucans were analyzed using an enzymatic assay kit (Megazyme International Ireland Ltd., Wicklow, Ireland) following the manufacturer's instruction. Briefly, the subtraction of the free glucose in the sample (blank) from the total glucose after enzymatic breakdown of β-glucans using glucose oxidase gives an accurate measurement of β-glucan. The relative amount of β-glucans in the sample was measured as β-glucans/nonreducing sugars.
decreased digestive complaints including diarrhea, stomach pain, and inflammatory bowel conditions (Slavin, 2013). Despite the bioactivity of natural barley bran, it is possible that barley bran fermented using Bacillus spp. can be more effective in terms of bioavailability of active compounds (e.g., β-glucan) with bacteria-derived enzymatic action (Choi, Kim, Ra, & Suh, 2007; Zhu et al., 2010). Previous studies have shown that sigumjang, which consists of barley bran fermented with B. amyloliquefaciens, contained 7-times more β-glucans than that of naturally fermented barley bran (Jeong, Lee, Kim et al., 2016). β-glucans are a crucial compound to treat gastrointestinal tract disorders (Vetvicka, Vannucci, & Sima, 2015; Yoshizawa, Yokoyama, Nakano, & Nakamura, 2004). Furthermore, plant and microbial-origin β-glucans showed various bioactivities including anti-inflammatory action (Mikkelsen, Jespersen, Mehlsenb, Engelsen, & Frøkiær, 2014; Schwartz & Hadar, 2014; Du, Lin, Bian, & Xu, 2015). Fermented barley bran (FBB) is the main ingredient of sigumjang, a traditional fermented food in South Korea (Jeong, Lee, Yoon et al., 2016). FBB is used to treat gastroenteric disorders, including indigestion, stomach pain, and inflammatory bowel conditions in South Korea. Despite the potential bioactivities of FBB on stomach ulcers and use as a folk remedy, there have been no studies on the gastroprotective activity of FBB extracts. Therefore, the present work investigates whether pretreatment of β-glucans-enriched FBB extracts can attenuate alcoholinduced gastric damage in vivo.
2.3. Animals and treatments 2.3.1. Animals Twenty-four male Institute of Cancer Research (ICR) strain mice aged 6 wk were purchased from Orientbio (Seongnam, South Korea) and acclimated for 1 wk before the experiments. Mice were housed in an air-conditioned room with a 12-h light/dark cycle at 22 ± 2 °C and humidity at 65%. Each group was provided an AIN-76 diet (purchased from Orientbio) (Bieri, 1979) and water ad libitum. This study was reviewed and approved by the Institutional Animal Care and Use Committee of Yeungnam University (IACUC YU 2015–017) and the animals were cared for in accordance with the Yeungnam University guidelines for animal experiments. 2.3.2. In vivo model of EtOH-induced gastric ulcer and FBB treatments Absolute EtOH was purchased from Sigma-Aldrich. Lyophilized FBB powders were dissolved in sterile distilled water prior to treatment. The appropriate application volume of extracts of FBB was confirmed through a preliminary experiment. The 24 healthy male ICR mice were randomly assigned to the following 5 experimental groups: (1) vehicle control (distilled water), (2) FBB extracts (single treatment), (3) EtOH, (4) FBB extracts (single treatment) + EtOH and (5) FBB extracts (multiple treatments: once a day for 1 wk) + EtOH (n = 6 mice/group). FBB extracts was administrated intragastrically at a dose of 500 mg/kg body weight prior to the acute alcohol challenge. To induce acute gastric mucosal injury, mice were treated with a single oral dose of absolute EtOH (3.9 g/kg body weight) 1 h after treatment with FBB extracts (Jeon, Lee, Shin, Lim, & Shin, 2012). Animals were fasted overnight before acute EtOH exposure and all animals were sacrificed 1 h after receiving the EtOH treatment (Supplementary Fig. 2).
2. Materials and methods 2.1. Preparation of fermented barley bran extracts Barley bran was purchased commercially in the local market (Gyeongsan, South Korea). Fermented barley bran (FBB) was prepared according to a previous report (Jeong, Lee, Yoon et al., 2016) (Supplementary Fig. 1). Briefly, barely bran was fermented with B. amyloliquefaciens MFST KCCM11719P (Korean Culture Center of Microorganisms, Seoul, South Korea). Extracts of FBB were diluted 5-fold in distilled water and extracted at 65 °C for 24 h with gentle shaking. Water-soluble extracts of FBB were centrifuged at 5000 ×g for 20 min (Thermo Fisher Scientific, Waltham, MA, USA) and the supernatants were further fractionated using Microcon YM-3 and YM-100 filters (nominal cutoff of 3 and 100 kDa) (Millipore, Billerica, MA, USA) and then lyophilized (Labconco, Kansas City, MO, USA).
2.4. Collection of serum and tissue sample 2.2. Chemical analysis of FBB extracts All animals were anesthetized using carbon dioxide inhalation for blood sample collection 1 h after EtOH treatment. Blood was drawn under anesthesia and all mice were euthanized using exsanguination through cardiac puncture after bleeding. The blood samples were centrifuged at 800 ×g for 20 min, which was done within 1 h of collection and the serum stored at −80 °C for 24 h. The stomachs were dissected, opened along the greater curvature and washed with cold sodium phosphate-buffered saline (PBS). The stomachs were stretched on clean paper with the mucosal surface facing upward.
2.2.1. Total phenolic compounds Total phenolic content was determined according to a previously described method (Kim, Kim, Lee, & Han, 2016). FBB extracts (1 mg/ mL) were dissolved in 50% MeOH and a 10-fold diluted sample was mixed with 0.2 mL of Folin-Ciocalteu's reagent (Sigma-Aldrich Inc., St. Louis, MO, USA) and then allowed to stand at room temperature of 25 °C for 3 min, after which Na2CO3 (0.4 mL, 2%) was added to the mixture. After 1 h, absorbance of the mixture was measured using a spectrophotometer (UV-1240, Shimadzu Co., Kyoto, Japan) at 725 nm. The results are expressed as gallic acid equivalents.
2.5. Gastric lesion and histopathological analysis Gastric lesions were evaluated with photographs of hemorrhagic erosions in the stomach taken with a photometric digital camera (C5060, Olympus, Tokyo, Japan). The areas of damage (hemorrhagic gastritis) were measured using Image J image analysis software version 1.44 (NIH, Bethesda, MD, USA) (Jung, Won, & Jun, 2013). Histological examination was done on the stomach samples harvested from each group. The samples were fixed in 4% formalin, sequentially dehydrated with 70%, 80%, 95% and 100% EtOH for 10 min each, and then embedded in paraffin. Three μm thick stomach sections using a microtome (Leica, Wetzlar, Germany) were prepared and stained with hematoxylin and eosin (H&E) and examined under a light microscope (Leica) at
2.2.2. Total sugar and reducing sugar Biochemical quantification of total carbohydrates and reducing sugar was done using colorimetric assays as described previously (Kim & Han, 2012). Total sugar content (mg%) was measured using the phenol–sulfuric acid method in which 1 mL of diluted sample was mixed with 1 mL of 5% phenol reagent, and then 5 mL of sulfuric acid was added. After 30 min incubation, the absorbance was measured at 470 nm. Reducing sugar content was analyzed with 3, 5-dinitrosalicylic acid reagent. A calibration curve was prepared, and the results are shown as mg% D-glucose equivalents. Non-reducing sugar content was estimated by subtracting the reducing sugars from total sugars. 21
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×200 magnification. Mucus thickness was measured using image analysis using the Bio Doc-It ™ system (UVP Inc., Upland, CA, USA).
Table 1 Contents of phenolic compounds and β-glucans in FBB and BB extracts.
2.6. Biochemical analyses BBEc FBBEd
2.6.1. Alcohol content in serum Alcohol content in the serum was determined using a quantitative EtOH assay kit (Roche, R-Biopharm GmbH, Darmstadt, Germany), according to the manufacturer's instructions. Briefly, 60 μL of serum was mixed with 1 mL of reaction mixture in a cuvette. After mixing for 3 min, the absorbance (A1) was measured at 340 nm. A second reagent was added and the mixture was incubated for an additional 3 min. The second absorbance (A2) was measured at the same wavelength. During the entire experiment, the cuvette was capped to prevent alcohol evaporation. The concentration of EtOH was calculated according to the equation provided with the kit. Alcohol concentration (g/L) = ((V × MW)/(ε × d × v × 2 ×1000)) × ΔA. Where V, final volume (mL); MW, molecular weight of EtOH (46.07 g/mol); ε, extinction coefficient of NADH at 340 nm (6.3/mmol/ cm); d, light path (1 cm); v, sample volume (mL); ΔA, A2-A1.
Total phenolic compounds (GAEa μg/ mL)
β-glucans/non-reducing sugarsb (%)
38.2 ± 0.3 38.5 ± 0.4
2.61 10.3**
Data are expressed as average of 3 independent measurement. a Gallic acid equivalent. b Non-reducing sugar was calculated by subtraction of total sugar by reducing sugar. c Barley bran extracts. d Fermented barley bran extracts. ** p < 0.01 versus BBE.
using the Student's t-test. Differences between groups were considered statistically significant when p < 0.05. The authors have also chosen to use 0.01 or 0.001 for some of the data to indicate the greater significance of the differences. 3. Results and discussion
2.6.2. Alcohol dehydrogenase (ADH) activity The activity of serum ADH was determined using Bostian's method with some modifications (Bostian & Betts, 1978). The reaction mixture consisted of 1.4 mL distilled water, 0.75 mL of 1.0 M Tris-HCl buffer (pH 8.8), 0.3 mL of 20 mM NAD+, and 0.3 mL of EtOH. The reaction mixture was pre-incubated with 0.15 mL of serum for 5 min at 30 °C and the change in absorbance at 340 nm was monitored for 5 min to determine the amount of NADH generated. ADH activity was shown as the relative ratio to vehicle control (normal, defined as 1).
3.1. Enrichment of β-glucan in FBB The influence of fermentation on barley bran was measured using chemical compositional analyses including total phenolics and β-glucans (Table 1). FBB extracts showed the increased amount of β-glucans among the non-reducing sugars while the content of phenolic compound was not significantly different with barley bran. The content of β-glucans normalized to non-reducing sugar in FBB extracts was 3.9fold higher than that of non-fermented barley bran extracts (Table 1). It is consistent with previous studies indicating the microbial fermentation can increase the extractability of bioactive compounds in food (Choi et al., 2007; Curiel et al., 2015; Limón et al., 2015; Zhu et al., 2010).
2.6.3. Quantification of TNF-α in serum Serum TNF-α concentration was measured using a Multi-Analyte ELISArray kit (SABiosciences, Frederick, MD, USA) according to the manufacturer's instructions. Briefly, 50 μL of serum was incubated in a 96-well plate for 1 h at room temperature and subsequently washed three times with TPBS (phosphate-buffered saline + 0.05% Tween 20) (Sigma-Aldrich). Next, antibody was added, followed by incubation at room temperature for 1 h and washing three times with TPBS. The color was developed using an avidin-horseradish peroxidase for 30 min, after which absorbance was measured using an enzyme-linked immunosorbent assay reader at 450 nm (SPARK, Tecan, Vienna, Austria). TNF-α concentration was determined from the standard curve and shown as the relative ratio to vehicle control (normal, defined as 1).
3.2. FBB extracts reduces acute gastric lesions induced by EtOH administration Since excessive consumption of alcohol could affect the development of gastritis (Ströhle, Wolters, & Hahn, 2012), the authors examine whether pretreatment of FBB extracts can reduce the bleeding of the stomach lining. As expected, neither EtOH exposure nor FBB extracts as a single treatment affected the bleeding of the stomach lining (Fig. 1A(a), (b)). However, in contrast, the EtOH treatment induced severe hemorrhagic longitudinal linear lesions with hyperemia in the gastric body of the stomach (Fig. 1A(c)) since alcohol is a toxin that irritates the stomach lining, causing it to become inflamed. However, pretreatment with FBB extracts as a single or multi-treatment significantly reduced EtOH-induced gastric damage and gastrointestinal bleeding (Fig. 1A(d), (e)). The measurement of gastric hemorrhagic area, an index for gastric damage, showed that EtOH administration induced severe gastric damage (3.7-fold increased vs. normal), while it was significantly reduced using FBB extract pretreatment showing 41% and 68% decrease in single and multiple treatments, respectively (Fig. 1B). These results are consistent with previous reports demonstrating β-glucans prevent the damage of gastric mucosa induced by acetylsalicylic acid (Ozkan et al., 2010).
2.7. Reverse transcriptase polymerase chain reaction (RT-PCR) Semi-quantitative RT-PCR was done using the method of Fan, Kim, and Han (2009) to determine the total RNA of the stomachs. Five μg of total RNA were used for first-strand cDNA synthesis. Aliquots of cDNA were amplified using the following primers: TNF-α, sense 5′-GACTCA GGATGCTACTGTTGC-3′ and antisense 5′-AGATAGCAAATCGGCTGA CGG-3′ (359 bp product); and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), sense 5′-CTCTACCCACGGCAAGTTCAA-3′ and antisense 5′-GGATGACCTTGCCCACAGC-3′ (515 bp product). Then RTPCR products were looked at on 1.0% agarose gel electrophoresis with the agarose gel containing a final ethidium bromide concentration of 0.1 μg/mL (Mupid-2plus, Advance, Kasugai, Japan). Band intensities were determined by image analysis using the Bio Doc-It ™ system. 2.8. Statistical Analysis
3.3. FBB extracts ameliorates the histopathological alterations induced by EtOH absorption
Data from the individual experiments are indicated as mean ± standard deviation (SD). All statistical analyses were done using the Statistical Package for the Social Sciences version 18.0 for Windows software (SPSS, Chicago, IL, USA). Treatment effects were analyzed
The animal model of EtOH-induced gastric damage has been widely used for evaluating the gastro-protective activities of drugs (Robert, Nezamis, Lancaster, & Hanchar, 1979). An acute EtOH challenge destroys the mucosal barrier, leading to the depletion of gastric wall 22
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Fig. 1. Protective effct of FBB extracts on EtOH-induced bleeding in the stomach. (A) Representative images showing the gross appearance of the area of gastric mucosa damage in mice. (a) Normal, vehicle control; (b) FBBE, single pretreatment of FBB extracts (500 mg/kg body weight); (c) Control, EtOH treatment (3.9 g/kg body weight); (d) FBBE(S), single pretreatment of FBB extracts (500 mg/kg body weight) (1 h before) prior to EtOH administration; (e) FBBE(M), multiplepretreatment (once a day for 1 wk) of FBB extracts (500 mg/kg body weight) prior to EtOH administration. (B) Histological damage index shown as gastrointestinal bleeding area (%) was quantified using Image J software. FBBE, fermented barley bran extracts. Data represent means ± SD (n = 6). Values are significantly different from each other at *p < 0.05, ***p < 0.001.
Fig. 2. Influence of FBB extracts treatment on EtOH-induced histological changes. Representative histomorphological appearance of the gastric mucosal layer in an ulcerated condition induced by EtOH. (a) Normal, vehicle control; (b) FBBE, single pretreatment of FBB extracts (500 mg/kg body weight); (c) Control, EtOH treatment (3.9 g/kg body weight); (d) FBBE(S), single pretreatment of FBB extracts (500 mg/kg body weight) (1 h before) prior to EtOH administration; (e) FBBE(M), multiple-pretreatment (once a day for 1 wk) of FBB extracts (500 mg/kg body weight) prior to EtOH administration. Quantification of histological damage index by changes in thickness of stomach wall and mucosa compared with normal condition. FBBE, fermented barley bran extracts. Data represent means ± SD (n = 6). Values are significantly different from each other at *p < 0.05, **p < 0.01.
FBB extracts treatment without EtOH exposure (Fig. 2A(b)). The incidence and severity of gastric histopathological lesions decreased in the single and multi-treatment of FBB extracts compared with that in the EtOH group (Fig. 2A(d) and (e)). These results were confirmed by the quantitative analysis obtained after measuring the thickness of gastric mucosa in mice (Fig. 2B). The thickness of the gastric mucosa in
mucus (Lee et al., 2015; Shin, Jeon, Shin, Cha, & Lee, 2013). For example, gastric mucosa of EtOH-treated mice showed wide-ranging histopathological changes, characterized by severe hemorrhage, loss of epithelial cells, and decreased mucosal thickness (Fig. 2A(c)). However, the normal group showed normal gastric mucosal architecture (Fig. 2A(a)). In addition, there were no histopathological changes in 23
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bloodstream may influence the measurement. Pretreatment of FBB extracts had a minimal effect on the oxidative reaction pathway modulating ADH activity. Therefore, the decreased EtOH concentration in serum is probably due to inhibited EtOH absorption through the gastric mucosa, which has less morphological alteration upon the pretreatment of FBB extracts. In addition, β-glucan in barley bran is acid resistant and thus it is stable when it passes through the stomach (Rahar, Swami, Nagpal, Nagpal, & Singh, 2011). Also, it is noted that soluble β-glucans can increase gastric retention time (De Vries, Gerrits, Kabel, Vasanthan, & Zijlstra, 2016). β-Glucan interacts with the β-glucan receptor (e.g., Dectin-1, as a lectin consisting complex) for its recognition in macrophages, neutrophil lineages, dendritic cells and T-cells in the gastric mucosa (Schwartz & Hadar, 2014; Willment, Gordon, & Brown, 2001). Hence, it is possible that certain domains of the mucous lining of the stomach can directly bind FBB extracts-derived β-glucans and subsequently modulate the impact of EtOH administration. Moreover, watersoluble β-glucans form gel-like physical structures which, in turn, can cover and penetrate the mucosal layer of the gastrointestinal tract. This leads to improvement of healing processes as tissues are protected (Gao et al., 2012). 3.5. FBB extracts inhibits the expression of pro-inflammatory mediators induced by EtOH exposure The inflammatory process is a major component of the mucosal defense against exogenous and endogenous factors in the gastrointestinal tract (Martin & Wallace, 2006). The inflammatory response can lead to mucosal injury and impaired healing processes. Especially, TNF-α as one of the pleiotropic inflammatory cytokines has a key role in inflammation-mediating tissue injury (Wise & Yao, 2003) and the production of this proinflammatory cytokine is also a key mediator of EtOH-induced gastric lesions (Choi, Hwang, & Nam, 2010). In parallel, the data showed that EtOH treatment significantly increased enhanced TNF-α mRNA expression in the stomach and consequently increased TNF-α level in the serum (Fig. 4). On the other hand, pretreatment with FBB extracts significantly inhibited TNF-α gene expression and secretion of the protein into the bloodstream; particularly the multiple-dose is which significantly altered gene expression (68% decrease vs. control) and detection of TNF-α (over 20% lower than control) (p < 0.001). These results indicated that the reduction of pro-inflammatory cytokines is one of the major factors involved in the gastroprotective action of FBB extracts on EtOH-induced gastric ulcers. β-glucans of plant or microbial origin are known as anti-inflammatory compounds. They can modulate the inflammatory response as mucosal defense and repair processes. However, several types of inflammatory tissue injury are also mediated by reactive oxygen species (ROS) with EtOH exposure (Kim et al., 2016). Thus, further work will include the determination of the antioxidant properties of FBB extracts to neutralize ROS. Since β-glucans showed ROS scavenging capacity (Agostini et al., 2015), FBB extracts-derived β-glucans may help to diminish the greater generation of ROS and minimize cell damage. The authors also recognized the complexity of crude extracts of FBB. Although the effect of FBB extracts on the damage protection of stomach mucosa was evaluated, further analysis on the identification of other active molecules could show a multifaceted role of FBB extracts. It will be interesting to investigate how β-glucan can be retained on the stomach mucosa using tracking of fluorescent or radiolabeled β-glucan on the mucosa using an antibody. This analytical tool enables the visualization of localized β-glucans and quantification of retained β-glucans in the mucosa area (Dadachova & Casadevall, 2014; McCann, Carmona, Puri, Pagano, & Limper, 2005).
Fig. 3. Effect of FBB extracts treatment on alcohol absorption and metabolism. (A) Measurement of serum EtOH concentrations and (B) changes in alcohol dehydrogenase (ADH) activity. (a) Normal, vehicle control; (b) FBBE, single pretreatment of FBB extracts (500 mg/kg body weight); (c) Control, EtOH treatment (3.9 g/kg body weight); (d) FBBE(S), single pretreatment of FBB extracts (500 mg/kg body weight) (1 h before) prior to EtOH administration; (e) FBBE(M), multiple-pretreatment (once a day for 1 wk) of FBB extracts (500 mg/ kg body weight) prior to EtOH administration. FBBE, fermented barley bran extracts. Data represent means ± SD (n = 6). Values are significantly different from each other at ***p < 0.001.
the pretreatment of FBB extracts groups (single (792 µm)/multiple (884 µm)) was significantly higher (p < 0.01) than control (294 µm) in a comparison of each corresponding values. Strikingly, the thickness of mucosa with the treatment of FBB extracts was similar to that of normal mucosa (900 µm). In addition, multiple-dose pretreatment with FBB extracts had a shielding effect on gastric mucosa, reducing the severity of gastric histopathological lesions. These results suggested prolonging exposure of β-glucans-enriched FBB extracts may give protection against EtOH. 3.4. Pretreatment with FBB extracts inhibits EtOH concentration in serum Serum alcohol concentration was measured before and 1 h after oral EtOH administration. After 1 h of EtOH exposure, alcohol concentrations in the serum ranged from 0.193 to 3.62 g/L. The alcohol concentration of serum after single or multi-pretreatment with FBB extracts were significantly decreased compared with that of the control group (Fig. 3, p < 0.001). Alcohol metabolism proceeds using an oxidative pathway mediated by alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH), which convert alcohol into acetaldehyde and acetate simultaneously (Li et al., 2014). Blood alcohol generally decreased over time by ADH-mediated conversion into acetaldehyde, which is then converted to acetic acid through an oxidation reaction (Bosron & Li, 1986). Hence, serum ADH activity was measured to determine whether FBB extracts increased the activity of enzymes involved in alcohol metabolism. Although both FBB extracts pretreatments dramatically decreased serum alcohol concentration, there were no significant changes of ADH activity in serum (Fig. 3B). However, it is possible that limited secretion of ADH from stomach/liver to the
4. Conclusion β-Glucans-enriched FBB extracts prevented gastric mucosal injury induced by acute alcohol intake. Pretreatment of FBB extracts, even for 24
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Journal of Digital Information Management, 11, 365–370. Kim, D., & Han, G. D. (2012). High hydrostatic pressure treatment combined with enzymes increases the extractability and bioactivity of fermented rice bran. Innovative Food Science and Emerging Technology, 16, 191–197. Kim, D., Kim, G. W., Lee, S. H., & Han, G. D. (2016). Ligularia fischeri extracts attenuates liver damage induced by chronic alcohol intake. Pharmaceutical Biology, 54, 1465–1473. Lee, I. C., Baek, H. S., Kim, S. H., Moon, C., Park, S. H., Kim, S. H., ... Kim, J. C. (2015). Effect of diallyl disulfide on acute gastric mucosal damage induced by alcohol in rats. Human and Experimental Toxicology, 34, 227–239. Li, S., Gan, L. Q., Li, S. K., Zheng, J. C., Xu, D. P., & Li, H. B. (2014). Effects of herbal infusions, tea and carbonated beverages on alcohol dehydrogenase and aldehyde dehydrogenase activity. Food & Function, 5, 42–49. Limón, R. I., Peñas, E., Torino, M. I., Martínez-Villaluenga, C., Dueñas, M., & Frias, J. (2015). Fermentation enhances the content of bioactive compounds in kidney bean extracts. Food Chemistry, 172, 343–352. Liu, Y. D., Liu, W., & Liu, Z. (2013). Influence of long-term drinking alcohol on the cytokines in the rats with endogenous and exogenous lung injury. European Review for Medical and Pharmacological Sciences, 17, 403–409. Martin, G. R., & Wallace, J. L. (2006). Gastrointestinal inflammation: A central component of mucosal defense and repair. Experimental Biology and Medicine, 231, 130–137. McCann, F., Carmona, E., Puri, V., Pagano, R. E., & Limper, A. H. (2005). Macrophage internalization of fungal beta-glucans is not necessary for initiation of related inflammatory responses. Infection and Immunity, 73, 6340–6349. Mikkelsen, M. S., Jespersen, B. M., Mehlsenb, A., Engelsen, S. B., & Frøkiær, H. (2014). Cereal β-glucan immune modulating activity depends on the polymer fine structure. Food Research International, 62, 829–836. Ozkan, O. V., Ozturk, O. H., Aydin, M., Yilmaz, N., Yetim, I., Nacar, A., ... (2010). Effects of β-glucan pretreatment on acetylsalicylic acid-induced gastric damage: An experimental study in rats. Current Therapeutic Research, 71, 369–383.
Fig. 4. Changes in EtOH-induced inflammatory response by FBB extracts treatment. (A) RT-PCR analysis for the expression of TNF-α. (B) TNF-α level in the serum determined using enzyme-linked immunosorbent assay. FBBE, fermented barley bran extracts. Data represent means ± SD. Values are significantly different from each other at *p < 0.05, **p < 0.01, ***p < 0.001. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
a single-time exposure, reduced abnormal alcohol absorption into the bloodstream through the protection of gastro mucus and further reduced the inflammatory response by suppressing the pro-inflammatory cytokine TNF-α. This study provides information toward understanding the beneficial effects of FBB extracts on EtOH-induced gastric mucosal damage. The mechanism of action of FBB extracts on alcohol absorption using gastric mucosa needs to be evaluated. In addition, further studies are needed to elucidate the active compounds including linkage analysis of β-glucans in FBB. Acknowledgment This work was supported by the 2015 Yeungnam University Research Grant. Conflict of interest The authors declare that there is no conflict of interest. Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at doi:10.1016/j.fbio.2019.01.007. 25
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Pharmazeuten, 35, 281–292. Vetvicka, V., Vannucci, L. V., & Sima, P. (2015). Role of β-glucan in biology of gastrointestinal tract. Journal of Nature and Science, 1, e129. Wang, F., Li, Y., Zhang, Y. J., Zhou, Y., Li, S., & Li, H. B. (2016). Natural products for the prevention and treatment of hangover and alcohol use disorder. Molecules, 21, 64. Willment, J. A., Gordon, S., & Brown, G. D. (2001). Characterization of the human βglucan receptor and its alternatively spliced isoforms. Journal of Biological Chemistry, 276, 43818–43823. Wise, G. E., & Yao, S. (2003). Expression of tumour necrosis factor-alpha in the rat dental follicle. Archives of Oral Biology, 48, 47–54. World Health Organization (2014). Global status report on alcohol and health. Geneva, Switzerland: WHO. http://apps.who.int/iris/bitstream/10665/112736/1/ 9789240692763_eng.pdf. Yoshizawa, M., Yokoyama, K., Nakano, Y., & Nakamura, H. (2004). Protective effects of barley and its hydrolysates on gastric stress ulcer in rats. Yakugaku Zasshi, 124, 571–575. Zhu, Y. P., Yamaki, K., Yoshihashi, T., Kameyama, M. O., Li, X. T., Cheng, Y. Q., ... Li, L. T. (2010). Purification and identification of 1-deoxynojirimycin (DNJ) in okara fermented by Bacillus subtilis B2 from Chinese traditional food (Meitaoza). Journal of Agricultural and Food Chemistry, 58, 4097–4103.
Rahar, S. I., Swami, G., Nagpal, N., Nagpal, M. A., & Singh, G. S. (2011). Preparation, characterization, and biological properties of β-glucans. Journal of Advanced Pharmaceutical Technology & Research, 2, 94–103. Rehm, J., Mathers, C., Popova, S., Thavorncharoensap, M., Teerawattananon, Y., & Patra, J. (2009). Global burden of disease and injury and economic cost attributable to alcohol use and alcohol-use disorders. The Lancet, 373, 2223–2233. Robert, A., Nezamis, J. E., Lancaster, C., & Hanchar, A. J. (1979). Cytoprotection by prostaglandins in rats. Prevention of gastric necrosis produced by alcohol, HCl, NaOH, hypertonic NaCl, and thermal injury. Gastroenterology, 77, 433–443. Schwartz, B., & Hadar, Y. (2014). Possible mechanisms of action of mushroom-derived glucans on inflammatory bowel disease and associated cancer. Annals of Translational Medicine, 2, 19. Shin, I. S., Jeon, W. Y., Shin, H. K., Cha, S. W., & Lee, M. Y. (2013). Banhabaekchulchunma-tang, a traditional herbal formula attenuates absolute ethanol-induced gastric injury by enhancing the antioxidant status. BMC Complementary and Alternative Medicine, 13, 170. Slavin, J. (2013). Fiber and prebiotics: Mechanisms and health benefits. Nutrients, 5, 1417–1435. Ströhle, A., Wolters, M., & Hahn, A. (2012). Alcohol intake–a two-edged sword. Part 1: Metabolism and pathogenic effects of alcohol. Medizinische Monatsschrift Fur
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Food Bioscience journal homepage: www.elsevier.com/locate/fbio
β-Glucan-enriched fermented barley bran (Sigumjang meju) extracts attenuates gastric mucosal injury induced by acute alcohol intake in vivo
T
Hojeong Jeonga,1, Dongyeop Kima,b,1, Hyo Jin Songa, Soohyung Leea, Mihyung Kima, ⁎ Keuk-Jun Kimc, Gi Dong Hana, a
Department of Food Science and Technology, Yeungnam University, Gyeongsan, Republic of Korea School of Dental Medicine, University of Pennsylvania, Philadelphia, PA, USA c Department of Clinical Pathology, Daekyeung University, Gyeongsan, Republic of Korea b
A R T I C LE I N FO
A B S T R A C T
Keywords: Fermented barley bran Bacillus spp. β-glucans Gastric mucosal injury Alcohol Sigumjang
Fermented barley bran (FBB) using Bacillus spp. is the main ingredient of sigumjang, a traditional fermented food in South Korea. The aim of this study was to evaluate the gastro-protective effects of β-glucan-enriched FBB extracts in an experimental mouse model of the EtOH-induced gastric ulcer. The gastro-protective activity of FBB was determined using histopathological inspection, level of EtOH absorption from the gastrointestinal-tract and measurement of pro-inflammatory cytokine. Pretreatment of FBB showed less gastrointestinal bleeding and reduced EtOH absorption into the bloodstream with the gastric mucosa. Furthermore, FBB effectively reduced the EtOH-induced mRNA expression of tumor necrosis factor α in the gastric mucosa and its level in the blood. These results showed that FBB can prevent gastric mucosal damage induced by acute EtOH-administration in mice by reducing absorption of EtOH by gastric mucosa along with decreasing the inflammatory response. Altogether, this data provides useful information on how β-glucan-enriched food modulates the EtOH-induced gastric mucosal damage, and thus, it could lead to the development of formulations for protecting gastric mucosal injury with acute alcohol intake.
1. Introduction Alcohol abuse is a devastating illness that has a public impact in the general population (Holden, 1987). Alcohol abuse has many long-term effects that result in premature death, causing 5.9% of all deaths globally. It also increases the propensity for serious illness and accounts for 5.1% of the global burden of alcohol-attributable disease and injury (Rehm et al., 2009; World Health Organization, 2014). The gastrointestinal (GI) tract is the primary digestive organ system for alcohol metabolism and absorption into the bloodstream (Bode & Bode, 1997). The direct contact of alcohol with the mucosa can induce numerous metabolic and functional changes, leading to mucosal damage, such as acute GI bleeding and diarrhea (Bode & Bode, 1997). The consumption of excessive alcohol causes necrotic lesions in the gastric mucosa due to acute hemorrhagic lesions, mucosal edema, epithelial exfoliation, and inflammatory cell infiltration, which in turn, reduces defensive factors such as bicarbonate secretion and mucus production (Choi et al., 2016; Guslandi, 1987). Furthermore, excessive
consumption of alcohol is responsible for a wide range of diseases, including alcoholic hepatitis, cirrhosis and hepato-carcinoma, pancreatitis, cardiomyopathy, hypertension, stroke, and fetal alcohol syndrome (Wang et al., 2016). In alcoholic organ damage, inflammation leads to an increase in the levels of inflammatory cytokines such as tumor necrosis factor α (TNFα), transforming growth factor α (TGF-α), interleukin 6 (IL-6), and interleukin 10 (IL-10). In particular, acute alcohol intake results in high levels of TNF-α in the bloodstream, suggesting that this proinflammatory cytokine has an important role in the pathogenesis of alcohol-induced acute gastric damage (Liu, Liu, & Liu, 2013). Hence, gastric mucosal protecting approaches modulating alcohol absorption, and thereby limiting the risk of the inflammatory response induced using an excessive amount of alcohol could be a potentially effective treatment to minimize the damage of acute alcohol intake. Barley bran is recognized as an alternative or complementary medicine. For example, it has been used for lowering blood sugar, blood pressure and cholesterol (Idehen, Tang, & Sang, 2017). Furthermore, it
⁎
Correspondence to: Department of Food Science and Technology, College of Life and Applied Sciences, Yeungnam University, 280 Daehak-Ro, Gyongsan, Gyeongbuk 58541, Republic of Korea. E-mail address: [email protected] (G.D. Han). 1 These authors contribute equally as co-first author. https://doi.org/10.1016/j.fbio.2019.01.007 Received 10 April 2018; Received in revised form 4 January 2019; Accepted 12 January 2019 Available online 15 January 2019 2212-4292/ © 2019 Elsevier Ltd. All rights reserved.
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2.2.3. Quantification of water-soluble β-glucan Contents of low-molecular weight and water-soluble β-glucans were analyzed using an enzymatic assay kit (Megazyme International Ireland Ltd., Wicklow, Ireland) following the manufacturer's instruction. Briefly, the subtraction of the free glucose in the sample (blank) from the total glucose after enzymatic breakdown of β-glucans using glucose oxidase gives an accurate measurement of β-glucan. The relative amount of β-glucans in the sample was measured as β-glucans/nonreducing sugars.
decreased digestive complaints including diarrhea, stomach pain, and inflammatory bowel conditions (Slavin, 2013). Despite the bioactivity of natural barley bran, it is possible that barley bran fermented using Bacillus spp. can be more effective in terms of bioavailability of active compounds (e.g., β-glucan) with bacteria-derived enzymatic action (Choi, Kim, Ra, & Suh, 2007; Zhu et al., 2010). Previous studies have shown that sigumjang, which consists of barley bran fermented with B. amyloliquefaciens, contained 7-times more β-glucans than that of naturally fermented barley bran (Jeong, Lee, Kim et al., 2016). β-glucans are a crucial compound to treat gastrointestinal tract disorders (Vetvicka, Vannucci, & Sima, 2015; Yoshizawa, Yokoyama, Nakano, & Nakamura, 2004). Furthermore, plant and microbial-origin β-glucans showed various bioactivities including anti-inflammatory action (Mikkelsen, Jespersen, Mehlsenb, Engelsen, & Frøkiær, 2014; Schwartz & Hadar, 2014; Du, Lin, Bian, & Xu, 2015). Fermented barley bran (FBB) is the main ingredient of sigumjang, a traditional fermented food in South Korea (Jeong, Lee, Yoon et al., 2016). FBB is used to treat gastroenteric disorders, including indigestion, stomach pain, and inflammatory bowel conditions in South Korea. Despite the potential bioactivities of FBB on stomach ulcers and use as a folk remedy, there have been no studies on the gastroprotective activity of FBB extracts. Therefore, the present work investigates whether pretreatment of β-glucans-enriched FBB extracts can attenuate alcoholinduced gastric damage in vivo.
2.3. Animals and treatments 2.3.1. Animals Twenty-four male Institute of Cancer Research (ICR) strain mice aged 6 wk were purchased from Orientbio (Seongnam, South Korea) and acclimated for 1 wk before the experiments. Mice were housed in an air-conditioned room with a 12-h light/dark cycle at 22 ± 2 °C and humidity at 65%. Each group was provided an AIN-76 diet (purchased from Orientbio) (Bieri, 1979) and water ad libitum. This study was reviewed and approved by the Institutional Animal Care and Use Committee of Yeungnam University (IACUC YU 2015–017) and the animals were cared for in accordance with the Yeungnam University guidelines for animal experiments. 2.3.2. In vivo model of EtOH-induced gastric ulcer and FBB treatments Absolute EtOH was purchased from Sigma-Aldrich. Lyophilized FBB powders were dissolved in sterile distilled water prior to treatment. The appropriate application volume of extracts of FBB was confirmed through a preliminary experiment. The 24 healthy male ICR mice were randomly assigned to the following 5 experimental groups: (1) vehicle control (distilled water), (2) FBB extracts (single treatment), (3) EtOH, (4) FBB extracts (single treatment) + EtOH and (5) FBB extracts (multiple treatments: once a day for 1 wk) + EtOH (n = 6 mice/group). FBB extracts was administrated intragastrically at a dose of 500 mg/kg body weight prior to the acute alcohol challenge. To induce acute gastric mucosal injury, mice were treated with a single oral dose of absolute EtOH (3.9 g/kg body weight) 1 h after treatment with FBB extracts (Jeon, Lee, Shin, Lim, & Shin, 2012). Animals were fasted overnight before acute EtOH exposure and all animals were sacrificed 1 h after receiving the EtOH treatment (Supplementary Fig. 2).
2. Materials and methods 2.1. Preparation of fermented barley bran extracts Barley bran was purchased commercially in the local market (Gyeongsan, South Korea). Fermented barley bran (FBB) was prepared according to a previous report (Jeong, Lee, Yoon et al., 2016) (Supplementary Fig. 1). Briefly, barely bran was fermented with B. amyloliquefaciens MFST KCCM11719P (Korean Culture Center of Microorganisms, Seoul, South Korea). Extracts of FBB were diluted 5-fold in distilled water and extracted at 65 °C for 24 h with gentle shaking. Water-soluble extracts of FBB were centrifuged at 5000 ×g for 20 min (Thermo Fisher Scientific, Waltham, MA, USA) and the supernatants were further fractionated using Microcon YM-3 and YM-100 filters (nominal cutoff of 3 and 100 kDa) (Millipore, Billerica, MA, USA) and then lyophilized (Labconco, Kansas City, MO, USA).
2.4. Collection of serum and tissue sample 2.2. Chemical analysis of FBB extracts All animals were anesthetized using carbon dioxide inhalation for blood sample collection 1 h after EtOH treatment. Blood was drawn under anesthesia and all mice were euthanized using exsanguination through cardiac puncture after bleeding. The blood samples were centrifuged at 800 ×g for 20 min, which was done within 1 h of collection and the serum stored at −80 °C for 24 h. The stomachs were dissected, opened along the greater curvature and washed with cold sodium phosphate-buffered saline (PBS). The stomachs were stretched on clean paper with the mucosal surface facing upward.
2.2.1. Total phenolic compounds Total phenolic content was determined according to a previously described method (Kim, Kim, Lee, & Han, 2016). FBB extracts (1 mg/ mL) were dissolved in 50% MeOH and a 10-fold diluted sample was mixed with 0.2 mL of Folin-Ciocalteu's reagent (Sigma-Aldrich Inc., St. Louis, MO, USA) and then allowed to stand at room temperature of 25 °C for 3 min, after which Na2CO3 (0.4 mL, 2%) was added to the mixture. After 1 h, absorbance of the mixture was measured using a spectrophotometer (UV-1240, Shimadzu Co., Kyoto, Japan) at 725 nm. The results are expressed as gallic acid equivalents.
2.5. Gastric lesion and histopathological analysis Gastric lesions were evaluated with photographs of hemorrhagic erosions in the stomach taken with a photometric digital camera (C5060, Olympus, Tokyo, Japan). The areas of damage (hemorrhagic gastritis) were measured using Image J image analysis software version 1.44 (NIH, Bethesda, MD, USA) (Jung, Won, & Jun, 2013). Histological examination was done on the stomach samples harvested from each group. The samples were fixed in 4% formalin, sequentially dehydrated with 70%, 80%, 95% and 100% EtOH for 10 min each, and then embedded in paraffin. Three μm thick stomach sections using a microtome (Leica, Wetzlar, Germany) were prepared and stained with hematoxylin and eosin (H&E) and examined under a light microscope (Leica) at
2.2.2. Total sugar and reducing sugar Biochemical quantification of total carbohydrates and reducing sugar was done using colorimetric assays as described previously (Kim & Han, 2012). Total sugar content (mg%) was measured using the phenol–sulfuric acid method in which 1 mL of diluted sample was mixed with 1 mL of 5% phenol reagent, and then 5 mL of sulfuric acid was added. After 30 min incubation, the absorbance was measured at 470 nm. Reducing sugar content was analyzed with 3, 5-dinitrosalicylic acid reagent. A calibration curve was prepared, and the results are shown as mg% D-glucose equivalents. Non-reducing sugar content was estimated by subtracting the reducing sugars from total sugars. 21
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×200 magnification. Mucus thickness was measured using image analysis using the Bio Doc-It ™ system (UVP Inc., Upland, CA, USA).
Table 1 Contents of phenolic compounds and β-glucans in FBB and BB extracts.
2.6. Biochemical analyses BBEc FBBEd
2.6.1. Alcohol content in serum Alcohol content in the serum was determined using a quantitative EtOH assay kit (Roche, R-Biopharm GmbH, Darmstadt, Germany), according to the manufacturer's instructions. Briefly, 60 μL of serum was mixed with 1 mL of reaction mixture in a cuvette. After mixing for 3 min, the absorbance (A1) was measured at 340 nm. A second reagent was added and the mixture was incubated for an additional 3 min. The second absorbance (A2) was measured at the same wavelength. During the entire experiment, the cuvette was capped to prevent alcohol evaporation. The concentration of EtOH was calculated according to the equation provided with the kit. Alcohol concentration (g/L) = ((V × MW)/(ε × d × v × 2 ×1000)) × ΔA. Where V, final volume (mL); MW, molecular weight of EtOH (46.07 g/mol); ε, extinction coefficient of NADH at 340 nm (6.3/mmol/ cm); d, light path (1 cm); v, sample volume (mL); ΔA, A2-A1.
Total phenolic compounds (GAEa μg/ mL)
β-glucans/non-reducing sugarsb (%)
38.2 ± 0.3 38.5 ± 0.4
2.61 10.3**
Data are expressed as average of 3 independent measurement. a Gallic acid equivalent. b Non-reducing sugar was calculated by subtraction of total sugar by reducing sugar. c Barley bran extracts. d Fermented barley bran extracts. ** p < 0.01 versus BBE.
using the Student's t-test. Differences between groups were considered statistically significant when p < 0.05. The authors have also chosen to use 0.01 or 0.001 for some of the data to indicate the greater significance of the differences. 3. Results and discussion
2.6.2. Alcohol dehydrogenase (ADH) activity The activity of serum ADH was determined using Bostian's method with some modifications (Bostian & Betts, 1978). The reaction mixture consisted of 1.4 mL distilled water, 0.75 mL of 1.0 M Tris-HCl buffer (pH 8.8), 0.3 mL of 20 mM NAD+, and 0.3 mL of EtOH. The reaction mixture was pre-incubated with 0.15 mL of serum for 5 min at 30 °C and the change in absorbance at 340 nm was monitored for 5 min to determine the amount of NADH generated. ADH activity was shown as the relative ratio to vehicle control (normal, defined as 1).
3.1. Enrichment of β-glucan in FBB The influence of fermentation on barley bran was measured using chemical compositional analyses including total phenolics and β-glucans (Table 1). FBB extracts showed the increased amount of β-glucans among the non-reducing sugars while the content of phenolic compound was not significantly different with barley bran. The content of β-glucans normalized to non-reducing sugar in FBB extracts was 3.9fold higher than that of non-fermented barley bran extracts (Table 1). It is consistent with previous studies indicating the microbial fermentation can increase the extractability of bioactive compounds in food (Choi et al., 2007; Curiel et al., 2015; Limón et al., 2015; Zhu et al., 2010).
2.6.3. Quantification of TNF-α in serum Serum TNF-α concentration was measured using a Multi-Analyte ELISArray kit (SABiosciences, Frederick, MD, USA) according to the manufacturer's instructions. Briefly, 50 μL of serum was incubated in a 96-well plate for 1 h at room temperature and subsequently washed three times with TPBS (phosphate-buffered saline + 0.05% Tween 20) (Sigma-Aldrich). Next, antibody was added, followed by incubation at room temperature for 1 h and washing three times with TPBS. The color was developed using an avidin-horseradish peroxidase for 30 min, after which absorbance was measured using an enzyme-linked immunosorbent assay reader at 450 nm (SPARK, Tecan, Vienna, Austria). TNF-α concentration was determined from the standard curve and shown as the relative ratio to vehicle control (normal, defined as 1).
3.2. FBB extracts reduces acute gastric lesions induced by EtOH administration Since excessive consumption of alcohol could affect the development of gastritis (Ströhle, Wolters, & Hahn, 2012), the authors examine whether pretreatment of FBB extracts can reduce the bleeding of the stomach lining. As expected, neither EtOH exposure nor FBB extracts as a single treatment affected the bleeding of the stomach lining (Fig. 1A(a), (b)). However, in contrast, the EtOH treatment induced severe hemorrhagic longitudinal linear lesions with hyperemia in the gastric body of the stomach (Fig. 1A(c)) since alcohol is a toxin that irritates the stomach lining, causing it to become inflamed. However, pretreatment with FBB extracts as a single or multi-treatment significantly reduced EtOH-induced gastric damage and gastrointestinal bleeding (Fig. 1A(d), (e)). The measurement of gastric hemorrhagic area, an index for gastric damage, showed that EtOH administration induced severe gastric damage (3.7-fold increased vs. normal), while it was significantly reduced using FBB extract pretreatment showing 41% and 68% decrease in single and multiple treatments, respectively (Fig. 1B). These results are consistent with previous reports demonstrating β-glucans prevent the damage of gastric mucosa induced by acetylsalicylic acid (Ozkan et al., 2010).
2.7. Reverse transcriptase polymerase chain reaction (RT-PCR) Semi-quantitative RT-PCR was done using the method of Fan, Kim, and Han (2009) to determine the total RNA of the stomachs. Five μg of total RNA were used for first-strand cDNA synthesis. Aliquots of cDNA were amplified using the following primers: TNF-α, sense 5′-GACTCA GGATGCTACTGTTGC-3′ and antisense 5′-AGATAGCAAATCGGCTGA CGG-3′ (359 bp product); and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), sense 5′-CTCTACCCACGGCAAGTTCAA-3′ and antisense 5′-GGATGACCTTGCCCACAGC-3′ (515 bp product). Then RTPCR products were looked at on 1.0% agarose gel electrophoresis with the agarose gel containing a final ethidium bromide concentration of 0.1 μg/mL (Mupid-2plus, Advance, Kasugai, Japan). Band intensities were determined by image analysis using the Bio Doc-It ™ system. 2.8. Statistical Analysis
3.3. FBB extracts ameliorates the histopathological alterations induced by EtOH absorption
Data from the individual experiments are indicated as mean ± standard deviation (SD). All statistical analyses were done using the Statistical Package for the Social Sciences version 18.0 for Windows software (SPSS, Chicago, IL, USA). Treatment effects were analyzed
The animal model of EtOH-induced gastric damage has been widely used for evaluating the gastro-protective activities of drugs (Robert, Nezamis, Lancaster, & Hanchar, 1979). An acute EtOH challenge destroys the mucosal barrier, leading to the depletion of gastric wall 22
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Fig. 1. Protective effct of FBB extracts on EtOH-induced bleeding in the stomach. (A) Representative images showing the gross appearance of the area of gastric mucosa damage in mice. (a) Normal, vehicle control; (b) FBBE, single pretreatment of FBB extracts (500 mg/kg body weight); (c) Control, EtOH treatment (3.9 g/kg body weight); (d) FBBE(S), single pretreatment of FBB extracts (500 mg/kg body weight) (1 h before) prior to EtOH administration; (e) FBBE(M), multiplepretreatment (once a day for 1 wk) of FBB extracts (500 mg/kg body weight) prior to EtOH administration. (B) Histological damage index shown as gastrointestinal bleeding area (%) was quantified using Image J software. FBBE, fermented barley bran extracts. Data represent means ± SD (n = 6). Values are significantly different from each other at *p < 0.05, ***p < 0.001.
Fig. 2. Influence of FBB extracts treatment on EtOH-induced histological changes. Representative histomorphological appearance of the gastric mucosal layer in an ulcerated condition induced by EtOH. (a) Normal, vehicle control; (b) FBBE, single pretreatment of FBB extracts (500 mg/kg body weight); (c) Control, EtOH treatment (3.9 g/kg body weight); (d) FBBE(S), single pretreatment of FBB extracts (500 mg/kg body weight) (1 h before) prior to EtOH administration; (e) FBBE(M), multiple-pretreatment (once a day for 1 wk) of FBB extracts (500 mg/kg body weight) prior to EtOH administration. Quantification of histological damage index by changes in thickness of stomach wall and mucosa compared with normal condition. FBBE, fermented barley bran extracts. Data represent means ± SD (n = 6). Values are significantly different from each other at *p < 0.05, **p < 0.01.
FBB extracts treatment without EtOH exposure (Fig. 2A(b)). The incidence and severity of gastric histopathological lesions decreased in the single and multi-treatment of FBB extracts compared with that in the EtOH group (Fig. 2A(d) and (e)). These results were confirmed by the quantitative analysis obtained after measuring the thickness of gastric mucosa in mice (Fig. 2B). The thickness of the gastric mucosa in
mucus (Lee et al., 2015; Shin, Jeon, Shin, Cha, & Lee, 2013). For example, gastric mucosa of EtOH-treated mice showed wide-ranging histopathological changes, characterized by severe hemorrhage, loss of epithelial cells, and decreased mucosal thickness (Fig. 2A(c)). However, the normal group showed normal gastric mucosal architecture (Fig. 2A(a)). In addition, there were no histopathological changes in 23
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bloodstream may influence the measurement. Pretreatment of FBB extracts had a minimal effect on the oxidative reaction pathway modulating ADH activity. Therefore, the decreased EtOH concentration in serum is probably due to inhibited EtOH absorption through the gastric mucosa, which has less morphological alteration upon the pretreatment of FBB extracts. In addition, β-glucan in barley bran is acid resistant and thus it is stable when it passes through the stomach (Rahar, Swami, Nagpal, Nagpal, & Singh, 2011). Also, it is noted that soluble β-glucans can increase gastric retention time (De Vries, Gerrits, Kabel, Vasanthan, & Zijlstra, 2016). β-Glucan interacts with the β-glucan receptor (e.g., Dectin-1, as a lectin consisting complex) for its recognition in macrophages, neutrophil lineages, dendritic cells and T-cells in the gastric mucosa (Schwartz & Hadar, 2014; Willment, Gordon, & Brown, 2001). Hence, it is possible that certain domains of the mucous lining of the stomach can directly bind FBB extracts-derived β-glucans and subsequently modulate the impact of EtOH administration. Moreover, watersoluble β-glucans form gel-like physical structures which, in turn, can cover and penetrate the mucosal layer of the gastrointestinal tract. This leads to improvement of healing processes as tissues are protected (Gao et al., 2012). 3.5. FBB extracts inhibits the expression of pro-inflammatory mediators induced by EtOH exposure The inflammatory process is a major component of the mucosal defense against exogenous and endogenous factors in the gastrointestinal tract (Martin & Wallace, 2006). The inflammatory response can lead to mucosal injury and impaired healing processes. Especially, TNF-α as one of the pleiotropic inflammatory cytokines has a key role in inflammation-mediating tissue injury (Wise & Yao, 2003) and the production of this proinflammatory cytokine is also a key mediator of EtOH-induced gastric lesions (Choi, Hwang, & Nam, 2010). In parallel, the data showed that EtOH treatment significantly increased enhanced TNF-α mRNA expression in the stomach and consequently increased TNF-α level in the serum (Fig. 4). On the other hand, pretreatment with FBB extracts significantly inhibited TNF-α gene expression and secretion of the protein into the bloodstream; particularly the multiple-dose is which significantly altered gene expression (68% decrease vs. control) and detection of TNF-α (over 20% lower than control) (p < 0.001). These results indicated that the reduction of pro-inflammatory cytokines is one of the major factors involved in the gastroprotective action of FBB extracts on EtOH-induced gastric ulcers. β-glucans of plant or microbial origin are known as anti-inflammatory compounds. They can modulate the inflammatory response as mucosal defense and repair processes. However, several types of inflammatory tissue injury are also mediated by reactive oxygen species (ROS) with EtOH exposure (Kim et al., 2016). Thus, further work will include the determination of the antioxidant properties of FBB extracts to neutralize ROS. Since β-glucans showed ROS scavenging capacity (Agostini et al., 2015), FBB extracts-derived β-glucans may help to diminish the greater generation of ROS and minimize cell damage. The authors also recognized the complexity of crude extracts of FBB. Although the effect of FBB extracts on the damage protection of stomach mucosa was evaluated, further analysis on the identification of other active molecules could show a multifaceted role of FBB extracts. It will be interesting to investigate how β-glucan can be retained on the stomach mucosa using tracking of fluorescent or radiolabeled β-glucan on the mucosa using an antibody. This analytical tool enables the visualization of localized β-glucans and quantification of retained β-glucans in the mucosa area (Dadachova & Casadevall, 2014; McCann, Carmona, Puri, Pagano, & Limper, 2005).
Fig. 3. Effect of FBB extracts treatment on alcohol absorption and metabolism. (A) Measurement of serum EtOH concentrations and (B) changes in alcohol dehydrogenase (ADH) activity. (a) Normal, vehicle control; (b) FBBE, single pretreatment of FBB extracts (500 mg/kg body weight); (c) Control, EtOH treatment (3.9 g/kg body weight); (d) FBBE(S), single pretreatment of FBB extracts (500 mg/kg body weight) (1 h before) prior to EtOH administration; (e) FBBE(M), multiple-pretreatment (once a day for 1 wk) of FBB extracts (500 mg/ kg body weight) prior to EtOH administration. FBBE, fermented barley bran extracts. Data represent means ± SD (n = 6). Values are significantly different from each other at ***p < 0.001.
the pretreatment of FBB extracts groups (single (792 µm)/multiple (884 µm)) was significantly higher (p < 0.01) than control (294 µm) in a comparison of each corresponding values. Strikingly, the thickness of mucosa with the treatment of FBB extracts was similar to that of normal mucosa (900 µm). In addition, multiple-dose pretreatment with FBB extracts had a shielding effect on gastric mucosa, reducing the severity of gastric histopathological lesions. These results suggested prolonging exposure of β-glucans-enriched FBB extracts may give protection against EtOH. 3.4. Pretreatment with FBB extracts inhibits EtOH concentration in serum Serum alcohol concentration was measured before and 1 h after oral EtOH administration. After 1 h of EtOH exposure, alcohol concentrations in the serum ranged from 0.193 to 3.62 g/L. The alcohol concentration of serum after single or multi-pretreatment with FBB extracts were significantly decreased compared with that of the control group (Fig. 3, p < 0.001). Alcohol metabolism proceeds using an oxidative pathway mediated by alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH), which convert alcohol into acetaldehyde and acetate simultaneously (Li et al., 2014). Blood alcohol generally decreased over time by ADH-mediated conversion into acetaldehyde, which is then converted to acetic acid through an oxidation reaction (Bosron & Li, 1986). Hence, serum ADH activity was measured to determine whether FBB extracts increased the activity of enzymes involved in alcohol metabolism. Although both FBB extracts pretreatments dramatically decreased serum alcohol concentration, there were no significant changes of ADH activity in serum (Fig. 3B). However, it is possible that limited secretion of ADH from stomach/liver to the
4. Conclusion β-Glucans-enriched FBB extracts prevented gastric mucosal injury induced by acute alcohol intake. Pretreatment of FBB extracts, even for 24
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Fig. 4. Changes in EtOH-induced inflammatory response by FBB extracts treatment. (A) RT-PCR analysis for the expression of TNF-α. (B) TNF-α level in the serum determined using enzyme-linked immunosorbent assay. FBBE, fermented barley bran extracts. Data represent means ± SD. Values are significantly different from each other at *p < 0.05, **p < 0.01, ***p < 0.001. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
a single-time exposure, reduced abnormal alcohol absorption into the bloodstream through the protection of gastro mucus and further reduced the inflammatory response by suppressing the pro-inflammatory cytokine TNF-α. This study provides information toward understanding the beneficial effects of FBB extracts on EtOH-induced gastric mucosal damage. The mechanism of action of FBB extracts on alcohol absorption using gastric mucosa needs to be evaluated. In addition, further studies are needed to elucidate the active compounds including linkage analysis of β-glucans in FBB. Acknowledgment This work was supported by the 2015 Yeungnam University Research Grant. Conflict of interest The authors declare that there is no conflict of interest. Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at doi:10.1016/j.fbio.2019.01.007. 25
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