Novel in vitro and in vivo anti-Helicobacter pylori effects of pomegranate peel ethanol extract

Background and Aim: Interest in plants with antimicrobial properties has been revived due to emerging problems associated with using antibiotics to eradicate Helicobacter pylori. Accordingly, this study aims to assess the antibacterial effects of Punica granatum and the possible synergistic effect of its extract along with metronidazole against H. pylori. Materials and Methods: Pomegranate peel ethanol extracts (PPEE) was tested against a control strain of H. pylori (NCTC 11916) in vitro and in vivo in female Wistar rats. Moreover, the synergistic effect of PPEE in combination with metronidazole was tested in vitro. Results: The PPEE exhibited a remarkable activity against H. pylori with a minimum inhibitory concentration (MIC) of 0.156 mg/mL. Furthermore, the extract exhibited a pronounced urease inhibitory activity (IC50 ~6 mg/mL) against the tested strain. A synergistic effect between PPEE and metronidazole was also observed (fractional inhibitory concentrations <0.5). Oral treatment of rats with PPEE for 8 days produced a significant reduction in H. pylori gastritis and a significant decrease in both lymphocytic and positive chronicity. Conclusion: Pomegranate extract is probably safe and represents a potential alternative and complementary therapy for reducing H. pylori associated with gastric ulcers.


Introduction
Peptic ulcer disease caused by Helicobacter pylori infection is a worldwide problem [1]. H. pylori can weaken the protective mucus coating of the stomach and the upper part of the small intestine, making them more vulnerable to the acidic gastric juices. This could lead to gastritis and ulcers. Other complications include chronic gastritis, gastric carcinoma, and primary gastric B-cell lymphoma [2].
Ancient civilizations used medicinal plants to treat a wide range of diseases. And in modern medicine, these plants represent a rich source for drug discovery [3]. Plant extracts contain bioactive compounds that can be used prophylactically and therapeutically over synthetic drugs [4,5] and may display potent antimicrobial properties [6]. Punica granatum or Pomegranate is one plant with great therapeutic potential. Pomegranate peel extract with its high antioxidant capacity exhibits anti-inflammatory and anti-ulcerogenic activities that are ten-fold higher than those of the pulp extract [7]. Moreover, research on the synergetic activity of pomegranate peel extract is sparse.
Accordingly, and as part of the Jordan traditional medicine project, the present study was designed to evaluate the anti-H. pylori and urease inhibitory activities of pomegranate peel ethanol extracts (PPEE) extract, and to investigate possible synergism between PPEE and metronidazole against this bacteria.

Ethical approval
This study was approved by the animal care committee at the University of Jordan.

Study period and location
This research was conducted for 8 months (between October 2015 and July 2016) at University of Jordan, Jordan.

Plant extraction
Peels of P. granatum (Pomegranate) were collected from North Jordan and identified by direct comparison with the authenticated sample at the herbarium of the Faculty of Science and the Faculty of Agriculture at The University of Jordan. A voucher specimen was deposited with a voucher number of Pun-001.
Peels were air-dried under ambient conditions for 7 days, then powdered using an electric grinder and stored in plastic bags. Approximately 500 g of finely-powdered peels were soaked in 70% EtOH in a closed container at room temperature at a ratio of 1:10 for 3 days. The pooled solvent was evaporated and produced a thick viscous pellet, which was further oven-dried at 50°C overnight, making a 27.77 g% yield w/w.

H. pylori isolation, culture media, and growth conditions
Twelve H. pylori clinical isolates and one reference strain of H. pylori (NCTC 11916) were used in this study. The clinical strains were isolated from gastric biopsy samples obtained by a gastroenterologist at the Jordan University Hospital during a routine endoscopy. Gastric biopsies were processed according to standard methodology [8]. Briefly, each biopsy was homogenized using a tissue homogenizer (IKA, Staufen, Germany). Aliquots of 100 µL of the homogenate were primarily cultured on Columbia blood agar (Oxoid, Hampshire, UK) supplemented with 7% (v/v) horse blood and Dent selective supplement (Oxoid, Hampshire, UK). The same bacterial plates were used for subcultures but without the Dent supplement. All plates were incubated at 37 °C under microaerophilic conditions using the CampyGen atmosphere generating system (Oxoid, Hampshire, UK) in anaerobic jars for 5-7 days. The growth of H. pylori was confirmed according to biochemical tests (positive for oxidase, catalase, and urease). H. pylori cultures were stored at −70°C in trypticase soy broth (Oxoid, Hampshire, UK) containing 10% (v/v) fetal calf serum (PAA, Pasching, Austria) and 15% glycerol [1,8]. All experiments were performed in triplicates to ensure consistency of the results.

Determination of H. pylori inhibition zones
The disk diffusion method was used to study the susceptibility of H. pylori to PPEE. The H. pylori suspension was prepared and adjusted to 6×10 8 CFU/mL (2 McFarland) in a fixed volume of 100 µL as described by Tayseer et al. [1] and Abu-Sini et al. [8]. The bacterial suspension was cultured on solid Columbia blood agar media. The PPEE stock solution was prepared by dissolving 2.5 g of the dry extract in a 2∶1 (v/v) DMSOphosphate-buffered solution (PBS) (pH 6.8) to obtain 2.5 w/v %. Thereafter, 30 µL of this extract were used to impregnate each of the sterile blank disks. These disks were left to dry, then placed on the H. pylori culture plates and incubated at 37°C for 1 h [9]. Disks with standard antibiotics (clarithromycin 15 µg, metronidazole 5 µg, ciprofloxacin 5 µg, and tetracycline 30 µg) were used as positive controls whereas, disks impregnated in a 2∶1 (v/v) DMSO-PBS (pH 6.8) only were used as negative controls [1]. Finally, zones of inhibition were measured and recorded.

Quantitative determination of urease inhibition concentration (IC 50 )
Urease inhibition assay was performed as described by Nagata et al. [11], where urease inhibition served as a proxy for H. pylori inhibition. A 10 µL of 6×10 8 CFU/mL H. pylori suspension was challenged with 200 µL of a detection reagent composed of 50 mM PBS (pH 6.8) containing 500 mM urea and 0.02% phenol red. Color development was monitored by measuring the O.D. at 555 nm wavelength in a 5-min interval. Samples of bacteria with a detection reagent only or PPEE with a detection reagent only served as controls. The following equation calculates the percentage of inhibition: % inhibition = ([activity without plant extract -activity with plant extract]/[activity without plant extract])×100.
A lack of potential urease inhibition was indicated if IC 50 was more than 12.5 mg/mL (inhibition of <60%). The IC 50 of PPEE was determined using ED 50 plus (v1.0) software (Vergas, 2000). The urease inhibitory activity of PPEE was compared to the reference urease inhibitor acetohydroxamic acid (AHA) from Sigma (USA).

Induction of H. pylori infection in vivo
Female Wistar rats were selected according to Crabtree et al. [12] selection criteria. Wistar rats were housed in the animal house of the University of Jordan, fed a commercial diet (Hammodeh factory, Jordan) ad libitum and given sterile tap water. A total of 45 8-weekold female Wistar-rats (197.7±1.38 g) were pre-treated with streptomycin suspended in tap water (5 mg/mL) for 3 days before the first H. pylori inoculation. After 1 day of fasting, rats were inoculated by gavage 1 mL/rat with H. pylori suspension of 9 McFarland (2.7×10 9 CFU/mL) twice daily at an interval of 4 h [13], and for 8 consecutive days. Control rats were inoculated with sterile PBS (non-infected group). Eight weeks after the last inoculation, rats were sacrificed, and their stomachs were removed and sliced into three parts. The antrum region was used for the urease test, the second part for bacterial colonization and inflammatory response by culture, and rapid urease test. Another part of the stomach was rinsed in PBS and homogenized using a tissue homogenizer (Germany). Thereafter, aliquots of 100 µL of the homogenate were cultured, as described above, following similar growth conditions. The remaining stomach part was fixed in 10% formalin. Samples were sent out to Alpha Medical for hematoxylin and eosin (H&E) staining and histopathological analysis. Updated Sydney System with visual scales was used as a reference standard grading system. The density of H. pylori indicated if the case is acute infection, chronic inflammation, intestinal metaplasia, or atrophy [3,14].
Infection with H. pylori was indicated if the rat met two or three of the following criteria: (a) A positive urease test of gastric biopsy; (b) a positive H. pylori growth in the cultured gastric biopsy; and/or (c) the presence of H. pylori curved bacilli in the gastric tissue by standard histological examination [13].

Acute toxicity test of pomegranate extract
Acute toxicity was evaluated in vivo in female Wistar rats. Tests were performed initially to ascertain the safety of the prepared doses of PPEE. According to the guidelines of the Organization for Economic and Cooperation and Development for testing of chemicals [15], three different doses of 50, 300, and 2000 mg/kg were administered orally through gastric gavage to three different groups of rats (n=3 per group) for the determination of the lethal dose that kills 50% of the test animals in a group (LD 50 ).
Rats were weighed before, during, and after the procedure to measure any changes in body weights. Rats were also monitored to observe any possible changes in their behaviors after dosing at least once during the first 30 min and periodically through 24 h. Special attention was given during the early 4 h and daily for 14 days. Rats were monitored to observe any signs of toxicity including changes in skin and eyes, breathing issue, seizures, or death.

Testing the effect of PPEE on H. pylori
Treatment with PPEE was initiated after 7 weeks of the last H. pylori inoculation. The treatment was prepared by dissolving 1250 mg of PPEE in 100 mL water. A PPEE dose 50 mg/kg was administered twice daily for 8 days. The treatment was chosen based on a published protocol by Moghaddam et al. [7]. The PPEE dose represents 1/40 of the highest safe dose tested in the acute toxicity study (2000 mg/kg).

Statistical analysis
Chi-square test was used to test the difference between rat groups. p<0.05 was set to indicate statistical significance. Microsoft Windows 7 Excel program was used to conduct the analysis.

Pomegranate peel extract exhibits anti-H. pylori activity in vitro
The antibacterial activity of PPEE against the control H. pylori strain of as represented by the inhibition zone was 32.53±0.33 mm, compared to 65 mm, 56 mm, and 14 mm of ciprofloxacin, tetracycline, and clarithromycin, respectively, while no H. pylori inhibition by metronidazole was observed (Table-1).
The average MIC values of PPEE against the clinical H. pylori strains were 0.163±0.04 mg/mL. In comparison, The MICs of ciprofloxacin and metronidazole (positive controls) were 0.00043±6.0×10 -5 and 0.442±0.035 mg/mL, respectively (Table-2). Furthermore, the MIC of PPEE against the control H. pylori strain was 0.156 mg/mL as compared to 0.0006 mg/mL for ciprofloxacin, while the strain was resistant to metronidazole with a MIC of 0.256 mg/mL. The IC 50 for urease inhibition by PPEE was 6.3 mg/mL.

Pomegranate peel extract and metronidazole exhibit synergistic activity against H. pylori
According to the standard checkerboard test, the MIC for PPEE was 0.156 mg/mL, and the MIC for PPEE in combination with metronidazole was Available at www.veterinaryworld.org/Vol.14/January-2021/16.pdf 0.039 mg/mL. The MIC for metronidazole was 0.256 mg/mL, and the MIC for metronidazole in combination with PPEE was 0.016 mg/mL. The FIC values and FIC index showed a synergic effect for the combination (<0.5), where metronidazole FIC value mean (0.0625), PPEE FIC value mean (0.25), and FIC index (0.313). This indicates that the combination of PPEE with metronidazole was synergic.
Besides, PPEE was fractionated by column chromatography using solvents differing in their polarity; hexane, chloroform, ethyl acetate, butanol, and water. Fractions were assessed for their potential anti-H. pylori activity using antimicrobial susceptibility tests and in vitro interaction between the isolated compounds and Metronidazole (Synergism). A total of 0.3 mg/disk of butanol fraction displayed the highest inhibition zone of 15.6±0.3 mm among other fractions. Moreover, we performed a phytochemical analysis of the butanol fraction, which revealed the presence of gallic acid, mannitol sugar, and 5-hydroxy methyl furfural. Gallic acid, the major component in this fraction showed weak anti-H. pylori activity. On the other hand, gallic acid did not show any synergistic activity with metronidazole. Furthermore, 5-hydroxy methyl furfural was not tested against H. pylori as it was present in very minute amount in the butanol extract.

Acute in vivo administration of pomegranate peel extract shows no signs of toxicity
During the 14 days of the experiment, even with a high PPEE dose of 2000 mg/kg, no significant change in rats' weight compared with the control group was noticed (p>0.05). Moreover, no signs of toxicity or deaths were reported. The LD 50 value was indeterminable even at the highest PPEE dose, which indicates that PPEE is non-toxic at doses of expected efficacy. Results show that the extract is non-toxic even at relatively high concentrations (Table-3). Based on these findings, the starting PPEE dose was selected at 1/40 th of 2000 mg/kg or 50 mg/kg.

Infected non-treated group
Gastric culture from all infected rats showed well-identified H. pylori colonies, positive Gram stain microscopic test, and positive catalase, and oxidase and urease activity tests. Specimens showed contamination with Candida, and other bacteria and spores and dysplasia were noticed in some infected stomachs (Figure-1). Bacterial urease activity was detected for each antral stomach part by developing an intense pink color for the urease reagent within 24 h (Figure-2). Histopathological analysis revealed H. pylori positivity in 100% of stomach specimens. Active infection was also found in 46.1% of the samples and positive chronicity in 84.6% (Figures-3-5, Table-4).

Infected treated group
Urease positivity was observed in 23% of the gastric samples. Moreover, 61.5% of histopathological specimens showed H. pylori positive gastric mucosa. Chronicity and infection activity were observed in 23% and 15.3% of rat specimens, respectively (Table-4).
Chi-square test indicated a significant difference (p<0.05) between infected treated and non-infected treated rats in terms of urease activity (Figure-2), treatment effects on eradicating H. pylori (Figure-3), the chronicity of gastric infection, and the percentage of infection activity ( Figures-4 and 5). This indicates that PPEE treatment displayed potent anti-H. pylori activity.

Non-infected treated group
Gastric culture showed no characteristic H. pylori Colonies in all samples. We observed Candida and other bacterial contamination. Moreover, 88.9% of gastric samples were urease negative, and histopathology results showed gastric mucosa with no H. pylori in 100% specimens. Chronicity and infection activity were found in 33.3% and 11.1% of the samples, respectively (Table-4).

Discussion
We demonstrated that pomegranate peel extract displays a significant inhibitory effect on H. pylori growth and urease enzyme activity. To the best of our knowledge, this is the first report on the potential inhibitory properties of PPEE against urease enzyme activity, highlighting a possible mechanism through which pomegranate peel extract inhibits H. pylori. Similar findings were reported using pomegranate peel methanol extract with an average inhibition zone of 39 mm at 0.1 mg/disk using the disc diffusion method [16]. Furthermore; higher MIC values of 0.625-0.780 mg/mL were reported [17]. The differences between our findings and the values reported in the literature can be attributed to the type of extract and the variability in the H. pylori strains used and their susceptibility or resistance to antimicrobial agents [18].
Combining PPEE with metronidazole reduced the MIC for this antibiotic by four-folds. This suggests that effective therapy may be achieved with this antibacterial combination. Interestingly, a study by Voravuthikunchai et al. [17] reported that Pomegranate fruit rind extract displays anti-adhesive effects against H. pylori to gastric mucosa by altering the bacterium's cell surface hydrophobicity, which may synergistically facilitate the elimination of bacterial cells from the human body. However, in our study, we did not investigate the mechanisms behind the synergistic activity between metronidazole and PPEE. Future studies must focus on this aspect.
The model used by Werawatganon emphasized the importance of CFU per dose, the number of inoculations, and the pretreatment with antibiotics. We used this model of H. pylori infection to mimic human mild chronic gastritis and study the efficacy of PPEE against H. pylori infection [13]. In our study, H. pylori colonization was successfully established in all H. pylori inoculated-rats with an infection rate of 100%. This was statistically significant compared to non-infected controls (p<0.05). Compared to the previous works on Wistar rats, Li et al. [19] reported successful colonization of H. pylori in rats and mild-to-moderate mucosal inflammation. In a rat model of chronic H. pylori infection, Werawatganon reported a 69.8-83.0% success rate of infection [13]. However, our model achieved a higher colonization rate than what was reported in the literature. A possible explanation of this could be attributed to the repeated inoculation of H. pylori during the protocol. Our data demonstrate that the protocol we adopted is not only reliable for establishing H. pylori infection in Wistar rats but also for conducting in vivo experiments for H. pylori eradication by herbal extracts. Thus, our modified rat model can be used to assess potential therapies for treating human H. pylori      [7] reported that the hydroalcoholic extract of Pomegranate fruit peel exhibits potential antiulcer properties in Wistar rats. Such efficacy was attributed to the high antioxidant activity of Pomegranate fruit extract [7]. Future studies may focus on exploring the bioactive ingredients in pomegranate peel that may be responsible for its anti-H. pylori effects. Matsubara et al. [20] found that the antioxidative flavonoid epigallocatechin gallate (the main component in green tea extracts) exhibits potent H. pylori urease inhibitory activity with an IC 50 value of lower than that of acetohydroxamic acid in vitro. Notably, the most abundant catechins in Pomegranate are the potent antioxidant epicatechin and epigallocatechin gallate [21]. This hints that the anti-H. pylori effects of pomegranate peel may at least in part be attributed to its catechins.
In our study, PPEE treatment led to a modest reduction in H. pylori colonization and infection in Wistar rats (~39% treatment rate, p<0.05) after 8 days of treatment with a PPEE dose of 50 mg/kg. The treatment also significantly reduced infection chronicity (p<0.05). Furthermore, the anti-inflammatory effect of PPEE on gastric mucosa was remarkable. Since PPEE demonstrated no signs of acute toxicity at much higher doses (2000 mg/kg), it would be interesting to test the potency of higher PPEE doses (>50 mg/kg) against H. pylori. This study is superior to other studies that used Pomegranate extracts for anti-ulcerogenic and anti-inflammatory activities in non-H. pylori models [6,7].   Available at www.veterinaryworld.org/Vol.14/January-2021/16.pdf

Conclusion
Pomegranate peel extract exhibited remarkable activity against H. pylori in vitro with a MIC of 0.156 mg/mL, and a pronounced urease inhibitory activity with an IC 50 of ~6 mg/mL against the tested strain. We found a synergic effect of PPEEmetronidazole combination against H. pylori with an FIC of <0.5. Oral treatment of rats with PPEE for 8 days led to a significant reduction in H. pylori gastritis, in addition to a significant decrease in both lymphocytic and positive chronicity. Importantly, PPEE showed no signs of acute in vivo toxicity even at relatively high doses. Therefore, we propose PPEE as a potential alternative or complementary therapy to treat H. pylori infection associated with clinical symptoms, but more investigations are needed.

Authors' Contributions
AM and MAS performed the experiments. AM wrote the paper. LA, RMD, and AK conceived and designed the microbiological and biological activities. TA and WZ designed the extracted solutions and their corresponding experiments. RA, RTA, and IH analyzed and interpreted the data. All authors have read and approved the final manuscript.