Protective effects of triple fermented barley extract (FBe) on indomethacin-induced gastric mucosal damage in rats

Sixty healthy male 6-week old Sprague-Dawley SD (SPF/VAF Outbred CrljOri:CD1) rats purchased from OrientBio Co., Seungnam, Republic of Korea, were used in this study following acclimatization for 10 days. Animals were raised in polycarbonate cages at 20–25 °C and a relative humidity of 30–35%. The rats were subjected to 12-h photoperiod while food and water was freely accessible. With six groups and eight rats in each group, 48 rats were selected based on the body weight (average 258.58 ± 15.62 g, ranged in 229.00–283.00 g) measured one day before test material administration and used for the experiments. Experimental groups (eight rats in each of the six groups) are presented in Fig. 1.

The animal experiments were conducted according to the international regulations of the usage and welfare of laboratory animals and were approved by the Institutional Animal Care and Use Committee, Daegu Haany University, Gyeongsan, Rep. of Korea [Approval No: DHU2014–087].

Nutritional facts, including calories, carbohydrates, proteins, lipids, and dietary fiber levels were evaluated, according to the method of Food Code [26]. Total polyphenols and total flavonoids were evaluated according to the method of Health Functional Food Code [27].

The triple fermented barley extract (FBe) was provided by Glucan Corporation (Busan, Rep. of Korea). The FBe extract was prepared in three different steps. In saccharification (the first step), 1 kg non-glutinous barley was washed, soaked (6 h), drained (30 min), and steamed (15 min at 121 °C). After cooling, malt solution [10 g malt powder in 4 L distilled water] was added to the mixture and fermented for 12 h at 55 °C in a 20 L glass bioreactor. In the second step, 20 mL suspension of Saccharomyces cerevisiae (ATCC, VA, USA) was added to the first fermentate, mixed thoroughly, and fermented again at 30 °C for 48 h. In the last step, 20 mL of Weissella cibaria (lactic acid bacteria; KACC, Suwon, Rep. of Korea) was added to the second fermentate, mixed thoroughly, and fermented third time at 30 °C for 48 h. The prepared third fermentate was sterilized (150 °C for 15 min) and sieved through a 40-mesh sieve to get the final product (triple fermented barley extract; FBe). Some specimens of FBe [Code: FBe2014Ku01] were deposited in the herbarium of the Medical Research Center for Globalization of Herbal Formulation, Daegu Haany University, Gyeongsan, Rep. of Korea. The final product was stored at 4 °C to protect from moisture and light. FBe solutions (100, 200, and 300 mg kg^− 1) were prepared by dissolving in distilled water (DW) and orally administered once (in a volume of 5 mL kg^− 1 of rat) 30 min before IND (25 mg kg^− 1 in a volume of 5 mL kg^− 1 of rat) administration. Omeprazole (10 mg kg^− 1; OM; Sigma-Aldrich, St. Louis, MO, USA) was used as a reference drug [26]. To provide the similar experimental settings and administration stress, IND and intact control rats were once orally administered with 5 mL kg^− 1 (v/w) of DW instead of the test material (Fig. 1).

Thirty minutes after administration of vehicle, dosages of FBe or OM on 24 h fasted rats, IND (Sigma-Aldrich) was single orally administered at a dosage of 25 mg kg^− 1 of rat [28]. In intact controls, only sterilized DW (5 mL kg^− 1) was administered once by gastric gavages instead of IND (Fig. 1).

The rats were sacrificed by cervical dislocation under anesthesia using 99.50% CO₂ gas 6 h after treatment with IND or vehicle. The stomach was excised after a median incision, fixed in 10% neutral buffered formalin for 24 h, and acquired digital images. The ulcer areas on the stomach surface were estimated by an automated image analyzer (IMT i-solution Inc., Quebec, Canada) as reported by Suleyman et al. [28]. Gastric damage scores were calculated macroscopically by measuring the visible lesions.

A 0.2 g of the tissue sample was homogenized in 10 volumes of ice-cold potassium phosphate buffer [50 mM K₂HPO₄, pH 6.0 + 0.5% hexadecyltrimethylammonium bromide (HETAB)], and centrifuged at 12,000×g for 10 min at 4 °C [29]. Supernatant was discarded and the pellet was re-suspended in equal volumes of potassium phosphate buffer and 10 mM EDTA. One unit of myeloperoxidase (MPO) activity was defined as the amount of MPO g^− 1 of tissue that changed an absorbance of 1.0 per min at 460 nm and 37 °C [30].

Thiobarbituric acid method was used to determine the concentration of gastric mucosal lipid peroxidation [31]. The corpus mucosa of stomach was scraped and homogenized in 10 mL of KCl solution (100 g L^− 1). Five hundred microliter of the homogenate was added into a new tube containing thiobarbituric acid solution [1.5 mL of 8 g L^− 1 2-thiobarbiturate (Sigma-Aldrich), 0.2 mL of 80 g L^− 1 SDS, 1.5 mL of 200 g L^− 1 acetic acid, and 0.3 mL of DW], heated at 98 °C for 1 h, cooled at room temperature and added 5 mL of n-butanol: pyridine (15:1; Sigma-Aldrich). The mixture was vortexed, centrifuged at 3000×g for 30 min, and measured the absorbance of the supernatant by UV/Vis spectrophotometer at 532 nm. The 1,1,3,3-tetramethoxypropane (Sigma-Aldrich) was used to draw the standard curve and the results were displayed as nM of MDA g^− 1 of wet tissue (nM/g tissue).

The GSH content was estimated by following a previously reported method of Sedlak and Lindsay [32]. The stomach’s mucosal surface was homogenized in 2 mL of Tris-HCl buffer [50 mM Tris HCl, 0.2 mM sucrose, and 20 mM EDTA; pH 7.5], precipitated with 0.1 mL of 25% trichloroacetic acid and centrifuged at 3500×g for 40 min at 4 °C to remove the precipitate. The GSH content was determined in the supernatant using 2-nitrobenzoic acid (Sigma-Aldrich) by measuring the absorbance at 412 nm and the results were expressed as nM/mg tissue.

The CAT activity of tissue was calculated by following a previously reported study of Evans and Diplock [33]. Rat gastric mucosal homogenate was diluted with buffer as described before. The CAT dilution (2 mL) was mixed with 1 mL of 30 mM H₂O₂ and the absorbance was measured at 240 nm. The CAT activity was expressed in mM/min/mg of tissue.

The SOD activity was examined by following the previously reported methods of Bashir et al. [34] with slight modifications. Briefly, gastric homogenate (15 μl) was mixed with ethanol (250 μl), chloroform (125 μl), and cold deionized water (450 μl). The homogenate mixture was then centrifuged at 8000×g for 2 min at 4 °C and the extract (500 μl) was added to the reaction mixture [16% Triton X-100 (100 μl), 0.9 mM nitroblue tetrazolium (250 μl), and 500 μl of 72.4 mM triscacodylate buffer with 3.5 mM diethylene pentaacetic acid (pH 8.2)], incubated for 5 min at 37 °C, added 10 μl of 9 mM pyrogallol and incubated again for 5 min at 37 °C. After stopping the reaction with 2 M formic buffer, absorbance was measured at 540 nm. The SOD activity was expressed in mM/min/mg of tissue.

Regions of individual stomach samples, the fundus between the cardiac and pylorus were crossly trimmed based on the lumen, fixed in 10% neutral buffered formalin for 24 h, and paraffin-embedded sections (3–4 μm) were prepared. Selected sections were examined under a light microscope after staining with hematoxylin and eosin. The histological profiles of changes in total thicknesses of fundic mucosa and individual cross trimmed fundus tissues were observed under a microscope at a resolution of 400 ×. Furthermore, fundus lesion invasive percentage was also calculated according to the eq. (1) and semiquantitative scoring was assigned as follows; 0 = normal intact mucosa, 1 = slight surface erosive damages, 2 = moderate mucosa damages and 3 = severe total mucosa damages, based on general and histomorphometric analysis [35]. The histopathologist was blinded to the group distribution during analyses.

The experimental data were expressed as mean ± standard deviation (S.D.) of 8 rats. The variance homogeneity was estimated by the Levene’s test [36]. In case of insignificant deviation, the significance between different pairs was analyzed by one-way ANOVA using LSD. In the case of significant deviation, a non-parametric comparison test (Kruskal-Wallis H test) was used to compare the groups. If a significant difference was observed by the Kruskal-Wallis H test, the significance between specific pairs was estimated by the Mann-Whitney U test. Statistical analyses were performed using SPSS ver. 14 (IBM-SPSS Inc., Chicago, IL, USA) and the results were considered statistically significant at p < 0.05 and p < 0.01. Furthermore, to understand the efficacy of the tested substances, the percent changes between IND, FBe and OM treated rats were calculated by Eqs. (2) and (3), respectively [37].

Nutritional facts including calories, carbohydrates, proteins, lipids, and dietary fiber were 385.3 kcal 100 g^− 1, 93.0, 3.1, 0.1, and 20.20%, respectively. Total polyphenols and total flavonoids contents were 3.66 mg g^− 1 and 0.31 mg g^− 1, respectively (Table 1).

Slightly insignificant restricted ulcerative lesions were observed in intact control rats while focal hemorrhagic ulcerative lesions in IND treated rats were grossly dispersed in the gastric mucosa. Marked inhibitions of the gross gastric damages were noticed in OM (10 mg kg^− 1) and FBe (100, 200, and 300 mg kg^− 1) treated rats. Accordingly, a significant increase in gastric mucosal gross lesion areas was noticed in IND control groups compared with intact control groups, however, mucosal lesions significantly decreased in a dose-dependent manner when treated with FBe (100, 200, and 300 mg kg^− 1) or OM (10 mg kg^− 1). An inhibitory effect on the increase in IND-induced gastric mucosal gross lesion areas was noticed in groups treated with FBe (200 mg kg^− 1) or OM (10 mg kg^− 1; Figs. 2 and 3).

IND control groups showed a noteworthy increase in gastric MPO content compared to the intact control groups. A significant and dose-dependent decrease in MPO content was observed in rats treated with FBe (100, 200, and 300 mg kg^− 1). Furthermore, increase in IND-induced gastric MPO content was suppressed after a single oral application of OM (10 mg kg^− 1) which was similar to the inhibition observed by treatment with FBe (200 mg kg^− 1; Fig. 4).

IND control rats showed increases in gastric lipid peroxidation compared to the intact vehicle control rats. Whereas, significant and dose-dependent decreases in lipid peroxidation were observed in FBe (100, 200, and 300 mg kg^− 1) administered rats. Furthermore, IND-induced gastric lipid peroxidation was significantly suppressed after a single oral application of OM (10 mg kg^− 1) which was similar to the inhibition observed by treatment with FBe (200 mg kg^− 1; Table 2).

Statistically significant decreases in endogenous antioxidants and gastric GSH levels were noticed in IND control rats compared to the intact control rats, but these decreases in GSH levels induced by treatment with IND were significantly suppressed after a single oral application of FBe (100, 200, and 300 mg kg^− 1) or OM (10 mg kg^− 1). FBe (200 mg kg^− 1) revealed inhibitory effect on the decrease in IND-induced gastric GSH levels which was similar to those observed by treatment with OM (10 mg kg^− 1; Table 2).

IND control rats showed significant (p < 0.01) increases in gastric CAT activity as compared to the intact control rats, but these elevations of CAT activities induced by treatment of IND were significantly suppressed after a single oral application of FBe (100, 200, and 300 mg kg^− 1) or OM (10 mg kg^− 1). FBe at a dosage of 200 mg kg^− 1revealed similar inhibitory effect on the increase in IND-induced gastric CAT activities compared to those observed by treatment with OM (10 mg kg^− 1; Table 2).

IND control groups showed significant decreases in gastric SOD activities compared to the intact vehicle control groups. However, FBe (100, 200, and 300 mg kg^− 1) treated rats showed significant (p < 0.01) and dose-dependent increases in SOD activities. Furthermore, the decrease in IND-induced gastric SOD activity was significantly suppressed after a single oral application of FBe (200 mg kg^− 1) or OM (10 mg kg^− 1; Table 2).

Severe desquamation of focal epithelium, focal extensive superficial epithelial damage, congestion/ hemorrhages, and necrosis of ulcerative lesions and gastric glands were observed following treatment with IND in rats. However, these microscopic ulcerative lesions were significantly suppressed by treatment with test materials. During histomorphometric and semiquantitative analyses, a significant increase in semiquantitative histological scores and percentage of invaded lesions, and a decrease in peri-ulcerative mucosa thickness were noticed in IND control rats compared to the intact vehicle control rats. But, these changes were significantly regulated by treatment with FBe (100, 200, and 300 mg kg^− 1). Furthermore, a significant decrease in semiquantitative histological scores and percentage of invaded lesions, and an increase in peri-ulcerative mucosa thickness were noticed in OM (10 mg kg^− 1) administered rats compared to the IND control rats. FBe (200 mg kg^− 1) revealed inhibitory effect on the IND-induced histopathological gastric mucosal damages which was similar to those noticed in rats treated with OM (10 mg kg^− 1; Table 3, Fig. 5).