The Antibacterial Activity of Mass Galla Chinesis et Camelliae Fermentata on Helicobacter pylori Infection

Mass Galla chinesis et camelliae Fermentata (Chinese gall leaven, CGL) was investigated for activities against Helicobacter pylori (H. pylori) both in vitro and in vivo. The agar dilution method and time-kill curves, as in vitro assays and an in vivo study using a Kunming mice model, were performed. CGL demonstrated a strong anti-Helicobacter pylori activity in vitro with the minimal inhibitory concentrations (MICs) against multiple H. pylori strains of 0.5~8 mg/ml and the decreasing trend time-kill curves when increasing CGL concentrations. H. pylori eradication rates in vivo were evaluated based on rapid urease test (RUT) and histopathologic criteria. Results revealed that the eradication rates in the CGL groups were 40% (4/10) in the high dosage group, 33% (4/11) in the medium dosage group, and 18% (2/11) in the low dosage group, with the difference between the high dosage and H. pylori control groups being significant (P = 0.035). The H. pylori colonization scores could be reduced partly by CGL. These in vivo results demonstrated that CGL in a rationally high dosage might be the most effective.


Introduction
The discovery of Helicobacter pylori (H. pylori) in the 1980s caused a revolutionary change in the clinical treatment of gastritis and peptic ulcer disease [1]. Since then, the eradication of H. pylori has been recommended for H. pylori-associated diseases, and triple therapy, consisting of two antibiotics and one proton-pump inhibitor (PPI), became the standard treatment strategy and showed satisfying curative effect with the eradication rates > 90% [2]. However, the increasing rate of antibiotic resistance has resulted in the declined efficacy of triple therapy, which is no longer appropriate to be the firstline treatment in China due to the fact that the eradication rates could only reach 74.5%, currently [3]. Since the failure of standard triple therapy, alternative strategies, such as sequential, concomitant, and bismuth-containing quadruple therapies, have been applied, achieving promising results, and the updated Chinese guidance has recommended bismuthcontaining quadruple therapy, composed of PPI, two antibiotics, and bismuth, as the primary clinical treatment, with the medication time extended to 10-14 days [4,5]. However, the administration of long-term antibiotics produces more side effects and lowers patient compliance, and the occurrence of antibiotic resistance is still expected to increase. Therefore, searching for natural antibacterial agents to cure the pathogen is of concern. One hope as an alternative to antibacterial agents is traditional medicine that can effectively control this infection and eradicate H. pylori but with fewer side effects than antibiotics. As a source of greater chemical and bioactive diversity than synthetic drugs, traditional medicines offer a potential alternative for treatment methods. It is also important to identify effective and safe ones for H. pylori infection.
The fermentation processing of traditional Chinese medicines (TCMs) originated from the ancient brewing technology and had a long history in China. Fermented TCMs (FTCMs) had long been widely applied among folks for disease prevention and treatment [6]. Mass Galla chinesis et camelliae Fermentata (Chinese gall leaven, CGL), as one of the FTCMs, has been used to treat digestive and some infectious diseases frequently. The fermentation preparation is with Galla chinensis (Galla rhois) as the raw material and 2

Evidence-Based Complementary and Alternative Medicine
Thea viridis as the fermentation substrate. Galla chinensis has been proven to be effective for several kinds of bacteria including H. pylori due to its active ingredient gallic acid. Galla rhois had long been shown to have the inhibitory effect on H. pylori growth and H. pylori urease in vitro [7], and its extract had demonstrated the effect to cure H. pylori infections and protect against H. pylori-induced pathology in a Mongolian gerbil model [8]. Gallic acid, as the active ingredient of Galla chinensis, also showed inhibitory effect on H. pylori strains and the effects were dependent on dose and contact time [9]. But, the further application of Galla chinensis is limited by the dyspepsia symptoms, which are caused by the excessive tannin ingredient [10]. The fermentation method can convert tannin to gallic acid, so the ingredients of CGL are more reasonable than Galla chinensis to human body. In this study, the anti-Helicobacter effects of CGL were investigated using in vitro time-kill curves and an in vivo study with a Kunming mice model. H. pylori growth-inhibitory activity of CGL was determined by the agar dilution MIC testing. The test was performed by adding serial dilutions of CGL to the Columbia blood agar plates with the final concentrations ranging from 8 to 0.25 mg/mL. H. pylori (NCTC11637, 26695 and 8 random clinical antibiotic-resistant strains; 3 × 10 8 CFU/mL) was inoculated into the agar plates and cultured microaerobically at 37 ∘ C for 3 days. The minimal concentration of CGL that completely inhibited the growth of H. pylori was identified as the MIC. The non-drug agar was used as a negative control. All experiments were conducted in duplicate.

Materials and Methods
The bactericidal effect of CGL was examined by the time-kill curve methodology against the standard strain H. pylori NCTC11637. The strain was grown in the Columbia blood agar plates and incubated for 3 days at 37 ∘ C, followed by collecting the bacterial colonies in Brucella broth for preparation. The assays were performed using a shaking incubator set at 37.5 ∘ C, and four groups were set up, which were the 0.5x MIC CGL group, the 1.0x MIC CGL group, the 2.0x MIC CGL group, and a blank control group (in drugfree medium). The initial H. pylori inoculum was adjusted to 1 × 10 6 CFU/mL; then 0.1 mL was extracted to be added to 90 mm plates, with the corresponding concentrations of CGL and Brucella broth containing fetal calf serum added previously. Each plate containing 10 mL of final volume was incubated under a microaerobic environment. Colony counts were determined at 0, 4, 8, and 24 h. At each time point, 0.5 mL of the mother liquid was removed from the plates and serial 10-fold dilutions were made in Brucella broth. 0.1 ml of the diluted solution was plated on Columbia blood agar plates and incubated at 37 ∘ C, and colonies were counted and averaged after 3 days, with bactericidal rate = (control colony count − medication colony count)/control colony count × 100%. In addition, the time-kill curves were drawn with a semilog plot. The assays were performed in duplicate.

Animal Experiments. Specific pathogen-free (SPF) male
Kunming mice (22-28 g) were obtained from the Department of Laboratory Animal Science (Beijing, China; Certification Number SCXK 2011-0012) and kept in an isolated SPF barrier environment (room temperature, 23 ∘ C ± 1 ∘ C; humidity, 55%± 5%; light/dark cycle, 12/12-hour), with free access to sterilized mouse feed and water. The experiment was performed on the basis of the Guide for Animal Experimentation of Peking University First Hospital and approved by the Experimental Animal Ethics Committee (Certification Number J201539). All the animals were allowed to acclimate for 1 week before the start of the experiment.
Sydney strain of H. pylori (strain SS1) was incubated on Columbia blood agar plates containing 8% defibrinated sheep blood at 37 ∘ C under a microaerobic environment for 2-3 days, and then the bacterial colonies were collected in brain-heart infusion broth for preparation. Mice ( = 65) were divided into two groups: H. pylori-uninfected group ( = 7) and H. pylori-infected group ( = 58). All the animals in the H. pylori-infected group were given 200 mg/kg cyclophosphamide by intraperitoneal injection and then inoculated six times at 2-day intervals with 0.4 mL of brain-heart infusion broth containing H. pylori (12 × 10 8 CFU/mL) by intragastric gavage after fasting for 24 hours. Two weeks after H. pylori inoculation, one mouse in the H. pylori-uninfected group and three mice in the H. pylori-infected group were randomly selected and killed. The stomach samples were obtained and Evidence-Based Complementary and Alternative Medicine 3 the H. pylori colonization was confirmed by both rapid urease test (RUT) and Warthin-starry (W-S) staining. After successfully establishing H. pylori-infected model, all surviving mice, 6 uninfected and 55 infected, were divided into the following six groups: blank control group (H. pylori-uninfected, = 6), H. pylori control group (H. pylori-infected, = 11), triple therapy (H. pylori-infected, = 11), and CGL (high, medium, and low dosage, H. pylori-infected, = 11 per group) groups. The triple therapy group was treated with a suspension of lansoprazole (12.33 mg/kg), clarithromycin (205.50 mg/kg), and metronidazole (164.40 mg/kg) daily for 1 week. The CGL groups were treated with high, medium, and low dosages of CGL daily for 4 weeks. Mice in the H. pylori group were treated with sterile water in the same volume. No intervention was given in the blank control group. The medium dosage that was equivalent to the clinical administration was 1.85 g/kg, while 3.7 g/kg was the high dosage and 0.925 g/kg was the low dosage. One mouse of the CGL-high dosage group died in the process of gavage administration due to the wrong operation. Four weeks after in vivo drug administration, all animals were killed to collect the stomachs, which were opened along the greater curvature and washed with 4 ∘ C PBS. Half of the gastric antrum was scraped to detect H. pylori colonization by RUT, and the rest was fixed in formalin and embedded in paraffin for histopathological observation. Eradication rates were determined by the results of RUT and W-S staining. The degree of H. pylori colonization was determined through histopathological observation and scored based on the visual analogue scales of the updated Sydney System [11]. The density of H. pylori was graded with the concepts of normal, mild, moderate, and marked, which were replaced with numbers "0," "1," "2," and "3," respectively, in this article.

Statistical Analysis.
Fisher's Exact Test (SPSS 21.0; SPSS Inc., Chicago, IL, USA) was used for categorical data. Kruskal-Wallis ANOVA test was used for measurement data and a Dunn-Bonferroni test for post hoc comparisons. Data are expressed as mean ± SD, when appropriate. < 0.05 was considered statistically significant.

In Vitro.
Antibiotic resistances of clinically isolated strains against MZ or CH were screened according to the Etest. The drug resistance situation against clinically common antibiotics is shown in Table 1. Table 2 showed that CGL was effective against both antibiotic-resistant and sensitive strains with MICs of 0.5-8 mg/mL, while the MICs against antibiotic-resistant strains were lower than those against sensitive strains (0.5-2 versus 2-4 mg/mL), suggesting that CGL might be more useful against antibiotic-resistant strains. Figure 1 showed the bactericidal activity of CGL against H. pylori NCTC11637 at different concentrations, and the effect increased gradually at each time point. The time-kill curves are shown in Figure 2. The results revealed that 0.5x MIC could achieve the complete elimination of H. pylori after 24 h and suggested a dose-effect relationship of CGL. Statistically significant differences could be observed between the blank

In Vivo.
The change in body weight during the treatment is shown in Figure 3. The mice had a weight loss in week 1 after H. pylori successfully colonized the stomach. After 4 weeks of treatment (CGL, 4 weeks; triple therapy, 1 week), the mice had a progressive weight gain, and CGL showed no obvious influence on the weight of mice. However, statistical differences were observed between the blank control and the high dosage groups in week 1 and week 4 and the blank control and the low dosage groups in week 1. The H. pylori eradication rates, which were determined by both RUT and histopathology, are presented in Table 3. The final eradication rates were 0 (0/11) in the H. pylori control group, 91% (10/11) in the triple therapy group, 40% (4/10) in the CGL-high dosage group, 33% (4/11) in the CGL-medium dosage group, and 18% (2/11) in the CGL-low dosage group. Significant differences  existed between the H. pylori control and triple therapy/CGLhigh dosage groups. Although the differences between the triple therapy and the CGL (high, medium, and low dosage) groups were significant ( = 0.024, = 0.024, and = 0.002), CGL showed a positive effect as a single drug. The H. pylori eradication rates of CGL (medium and low dosage) groups were greater than that of H. pylori control group; however, no significant differences were observed between them and thus a larger sample size will be needed. The H. pylori colonization scores are shown in Figure 4, with no bacteria colonization in the blank control group, "1" and "2" as the major degrees in the H. pylori control group, "0" in the triple therapy group, "0" and "1" in the CGL (high and medium dosage) groups, and "1" in the CGL-low dosage group. Statistically significant differences existed between  H. pylori control and blank control/triple therapy groups. However, no significant differences were detected between the H. pylori control and the CGL (high, medium, and low dosage) groups. Similarly, there were also no statistically significant differences between the CGL groups and blank control/triple therapy groups. Figure 5 shows the successful colonization of H. pylori in gastric mucosa after H. pylori inoculation.

Discussion
CGL has been utilized for the treatment of digestive system diseases with the effects of removing phlegm, helping   produce saliva, and relieving thirst according to the traditional Chinese medicine (TCM) theory. Clinical observations indicated a significant improvement in clinical symptom alleviation in patients with gastric diseases concerning H. pylori. Nevertheless, current studies focus on its preparation technology. The anti-H. pylori effect remained unclear.
Our study confirmed the antibacterial activities of CGL both in vivo and in vitro by means of time-kill curves and H. pylori-infected mice model. Partly due to the limited data and the overly conservative statistical methods we adopted, significant differences were not detected between some groups, 1.0x MIC and blank control group in time-kill curve at the time point of 24 h, for example. These results strengthened the evidence for its clinical application. At present, CGL was applied to solve the complicated syndromes according to TCM theory and its satisfying safety with fewer side effects. This study expended its usage to the field of H. pylori infection treatment.
Despite the promising results, the eradication of CGL in vivo was not as ideal as that in vitro, possibly due to the gastric acid and mucus-bicarbonate barrier of the stomach. In CGL in in vivo experiment, the medium dosage was equivalent to the clinical administration, while the high dosage was double. However, the high dosage of CGL was excessively thick when converted to the animal dosage, and this may contribute to the relatively low weight of mice in the high dosage group. In addition, the body weight of mice had a slight decrease in week 1 after H. pylori successfully colonized the stomach, probably due to the initial, acute H. pylori infection phase. Considering the effect that excessively thick drug may cause and the weight gain that happened later, CGL showed no obvious influence on the weight of mice.
From the above, we learned that CGL was able to be an antibacterial agent. In addition, CGL also could be a health-promoting medicine. Through the fermentation, CGL is significantly different from Galla chinensis in its chemical 6 Evidence-Based Complementary and Alternative Medicine component contents, accompanied with the increased gallic acid and reduced tannins [12]. Moderate tannins have anticancer effects [13], but foods rich in tannins are recognized as being of low nutritional value, taking into account the responsibilities for decreases in feed intake, growth rate, net metabolizable energy, feed efficiency, and protein digestibility [10]. Gallic acid has antimicrobial, anti-inflammation, and antioxidation effects [14][15][16][17]. A drug containing rich gallic acid and moderate tannins is the most reasonable to human body, and CGL meets the requirements. Thus, CGL not only can be an antibacterial agent but also has the potential to be a promising healthcare medicine. The results of the present study confirmed the anti-H. pylori activity of CGL, indicating that CGL at a rationally high dosage might be the most effective. However, H. pylori SS1 was chosen as the test strain in the in vivo study due to its stable colonization. Thus, the anti-H. pylori effects on drug-resistant isolates in vivo still need to be verified. Successfully establishing a stable drugresistant animal model is essential for mimicking the human environment and further research determining the influence of the gastric environment on the anti-H. pylori activity of CGL would be helpful for improving its curative effect. Additionally, a combination of natural medicines has the prospect of becoming a better way to eradicate H. pylori since separate application may be slightly inferior to the antibiotics, and further research needs to be done.