Fermented‐Rhus verniciflua extract ameliorate Helicobacter pylori eradication rate and gastritis

Abstract An antibacterial effect of fermented‐Rhus verniciflua extract (FRVE), an urushiol‐free extract fermented by Fomitella fraxinea, on Helicobacter pylori was evaluated in mice. Minimal inhibitory concentration of FRVE against H. pylori eradication was checked with serial dilution method in vitro. H. pylori infection‐induced mice were utilized to determine the effect of oral administration of FRVE with/without standard triple therapy (STT: metronidazole, omeprazole, and clarithromycin) on H. pylori colonization and gastric inflammation. H. pylori was clearly eradicated by FRVE at a concentration of ≥2 mg/ml in vitro. In animal study, FRVE at a concentration of ≥6 mg/ml significantly reduced colonized H. pylori grading (0.2 vs. 2.2, p < .01) and improved gastric inflammation (0.4 vs. 1.6, p < .01) compared to control. STT with FRVE (3 mg/ml) exerted synergistic effect on both H. pylori colonization grade (STT, 0.6 ± 0.9; FRVE, 1.4 ± 0.5; STT + FRVE, 0.8 ± 0.4) and gastric inflammation (STT, 0.4 ± 0.5; FRVE, 1.4 ± 0.5; STT + FRVE,1.0 ± 0.1) compared with single therapy (p < .01). H. pylori eradication rate of FRVE (6 mg/ml) was higher than that of STT (60% vs. 20%). FRVE has potential antibacterial activity against H. pylori infection and can be used as an additional therapy on STT.

1-2 weeks is used worldwide for treating H. pylori infection. It has a high eradication success rate of >80% (Gong et al., 2014). However, STT is associated with a number of side effects, including antibiotic resistance and relapse (Ermis & Senocak Tasci, 2015). Therefore, alternative agent with less serious side effects needs to be identified for the eradication of H. pylori and treatment of gastric disease.
Rhus verniciflua Stokes (R. verniciflua) is commonly known as the lacquer tree . R. verniciflua is traditionally used in oriental medicine for treating a variety of diseases. The plant is also consumed as an ingredient in sumac chicken and duck in Korea (Jeong et al., 2015). Urushiol is a major component in the sap of R.
verniciflua. It has anti-inflammatory, antimicrobial, and antioxidant effects in mice (Bang et al., 2014). However, clinical trials of urushiol are hampered by the fact that it is a causative agent of allergic contact dermatitis .
Fermented-Rhus verniciflua extract (FRVE) is an urushiol-free extract fermented by Fomitella fraxinea (Choi et al., 2007). In a recent study, three phenolic acids (gallic acid, protocatechuic acid, and 4-hydroxy benzoic acid) and four flavonoids (fustin, fisetin, sulfuretin, and butein) have been detected in FRVE, with gallic acid and fisetin being its major compounds (Choi et al., 2012). FRVE also has anticancer, antioxidant, and anti-inflammatory effects. It can protect mice against hepatosteatosis (Choi et al., 2012). Antibacterial effect of FRVE is reported in recent studies. FRVE has growth inhibitory effect on H. pylori . And a study of antibacterial activity of major constituents which are methyl gallate, fustin, and quercitrin showed antibacterial effect of fustin and methyl gallate (Jang et al., 2016). A recent study has reported that fisetin has antibacterial effect (Leotoing et al., 2013). Moreover, gallic acid has antioxidant, antiproliferative, and antitumorigenic activities (Daglia et al., 2014). However, few have been reported about its effect on H. pylori has not been reported yet.
The objective of the present study was to determine the minimal inhibitory concentration of urushiol-free FRVE for H. pylori eradication. Reduction of H. pylori and improvement of gastritis after oral administration of FRVE were also compared to those by STT in C57BL/6 mice.

MATERIAL S AND ME THODS
Fermented R. verniciflua extracts FRVE, a fermented extract of bark part of R. verniciflua, and its constituent compounds (Table 1) were provided by HS Choi at Rural Development Administration, Republic of Korea. FRVE comprises abundant fustin, fisetin, and gallic acid in MS. The method for preparing FRVE has been described by Choi previously (Choi et al., 2012).
To absorb moisture in the lacquer bark, water was immersed for 1 day and drained for 1 day. Take out the lacquer bark, transfer it to a 5 L mushroom cultivation bag, put on a filter, and sterilize for 12 to 100 min. The prepared F. fraxinea mycelium was inoculated, cultured for 30 days at 21℃, and then dried with hot air at 50℃ to prepare fermented lacquer. FRVE was manufactured in powder form for use in experiments. The concentration was calculated according to the solvent (distilled water).

Animals
All animals received humane care in this study. All procedures were conducted in accordance with National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. All procedures were approved by Institutional Animal Care and Use Committee of Hallym University College of Medicine (2013-131; 2014-71; 2015-21). Six- week-old specific pathogen-free male C57BL/6 mice were purchased from Dooyeol Biotech (Seoul, Korea). All mice were housed individually in steel microisolator cages at 22 ± 2°C with 12 hr/12 hr of light/dark cycle. They were provided free access to water and food throughout the experiment period. They were monitored daily. These mice were divided into the following groups ( Figure 1  Optical density of H. pylori suspension was adjusted to 10, corresponding to 6.7 × 10 9 CFU/ml (Navabi et al., 2013). For standard triple therapy treatment, 400 mg/kg metronidazole, 20 mg/kg omeprazole, and 250 mg/kg clarithromycin were mixed in drinking water (Loughlin et al., 2003). Standard triple therapy was administered five times per week.
In the second week, mice in the four groups were infected with H. pylori for 2 weeks. In the fourth week, mice in the HP + FRVE group were divided into the following five subgroups and treated with 3, 4, 6, 8, or 16 mg/ml of FRVE suspension. Treatment of the FRVE suspension was performed 5 times per week for 3 weeks. Mice in the HP + FRVE+triple group were treated with 3 mg/ml of FRVE suspension for 3 weeks. In the fifth week, mice in the triple groups were treated with standard triple therapy for 2 weeks. Mice were euthanized in the seventh week.

Minimal inhibitory concentrations of FRVEs and H. pylori eradication
FRVE at 2 to 4 mg/ml eradicated H. pylori. However, H. pylori proliferation was unaffected by 1 or 1.5 mg/ml of FRVE (Figure 2

Improvement in gastritis
Regarding inflammation improvement, inflammation in HP had a mean grade of 1.6 ± 0.5 (Figure 4). This finding was shown on an- However, it was significantly (p < .01) lower than that in HP group.
No gastric inflammation was detected in the control group without any treatment.

Inflammatory cytokine
After H. pylori infection for 2 weeks, TNF-α concentrations did not differ significantly among groups ( Figure 5). However, IL-1β concentration in subgroup of HP + FRVE 8 and 16 mg/ml and HP + triple group was significantly lower than that in HP group.

DISCUSS ION
In this study, the MIC of FRVE against H. pylori in vitro and the concentration that resulted in the greatest reduction in H. pylori grades and improvement of inflammation in vivo were investigated. Application of 2 mg/ml of FRVE was highly effective in vitro while 6 mg/ml of FRVE was highly effective animal study. In this study, FRVE contained higher level of fustin than other components (Table 1). Previous reports have demonstrated that fustin possesses cell proliferationinhibiting and antiviral activities (Kang et al., 2012). Results for this study revealed that fustin also had antibacterial effect against H.
Therefore, FRVE has been subjected to clinical trials for various diseases (Lee et al., 2011).

F I G U R E 6 Study summary
Green tea, muscadine grape skin, broccoli, and cranberry have been investigated in vitro and/or in vivo as potential treatments for H. pylori (Brown et al., 2011). However, these studies did not make use of animal models and their concentrations were higher than 2 mg/ml, the concentration of FRVE used in this work. Thus, FRVE could be used as anti-H. pylori agent at a low concentration for a short period.
Gastric IL-1β expression is associated with H. pylori infection while urushiol therapy can reduce its expression (Lee et al., 2010;Suk et al., 2011). In our study, IL-1β level follows previous reports.
However, TNF-α expression levels were not affected by FRVE. FRVE exhibited more potent activity against H. pylori compared to the standard triple therapy comprising two antibiotic agents and a proton pump inhibitor ( Figure 3). In addition, FRVE significantly reduced H. pylori-induced gastritis, although such effect was not due to altered IL-1β or TNF-α expression in mice. Furthermore, the standard triple therapy together with a low concentration of FRVE exerted synergistic effect. Therefore, FRVE might be used as a potential adjuvant therapeutic agent for treating H. pylori-induced gastric disease ( Figure 6).