Phytochemicals in Helicobacter pylori Infections: What Are We Doing Now?

In this critical review, plant sources used as effective antibacterial agents against Helicobacter pylori infections are carefully described. The main intrinsic bioactive molecules, responsible for the observed effects are also underlined and their corresponding modes of action specifically highlighted. In addition to traditional uses as herbal remedies, in vitro and in vivo studies focusing on plant extracts and isolated bioactive compounds with anti-H. pylori activity are also critically discussed. Lastly, special attention was also given to plant extracts with urease inhibitory effects, with emphasis on involved modes of action.


Introduction
Plant products, their enriched-derived extracts, and their isolated bioactive molecules have been increasingly studied due their renowned health attributes, largely used in folk medicine over centuries for multiple purposes [1][2][3][4][5][6][7][8][9]. Indeed, phytomedicine is garnering much attention among the medical and scientific communities [10][11][12]. Commercially available synthetic drugs have often been negatively pointed out due to their side effects and related toxicity [13]. In fact, the active molecules used in pharmaceutical formulation are formerly derived from bioactive molecules extracted from plants and other living organisms [14]. Also, a growing number of studies have progressively underlined the multiple bioactive properties conferred by plant formulations [15,16]. Specifically, the antimicrobial effects of multiple plant preparations have been progressively confirmed and supported by both in vitro and in vivo studies and clinical trials [17][18][19][20][21]. Thus, their lower costs, high effectiveness, bioavailability, bioefficacy, and few to no adverse effects have led to intensive research on this topic [22][23][24][25][26][27][28].
Among the various opportunistic infections, those caused by Helicobacter pylori, a human opportunistic pathogen, is attracting much attention [29]. In fact, it is widely recognized that this bacterium plays an important role in the etiology of peptic and gastric ulcers and even gastric cancers and gastric lymphomas [29]. About half of the worldwide population is colonized by this bacterium, but there are only about 20% who manifest clinical symptoms, which has been linked to the ability of some H. pylori strains to both adapt to host's immunological responses and to support an ever-changing gastric environment [29]. Relatedly, increasing rates of antibiotic-resistant H. pylori strains have been found, and therefore, the search for new eradication strategies and effective antibiotic therapies has become an issue of crucial importance [30]. Hence, research effort is focused on exploring plants as sources of anti-H. pylori agents.
Based on these findings, the present report aims to provide an extensive overview of Helicobacter pylori infections, namely describing its involvement in triggering gastric cancer and the most common antimicrobials used in H. pylori eradication. Special attention is also given to medicinal plants and their corresponding extracts and isolated constituents used as anti-H. pylori agents and urease inhibitors. This review was performed by consulting the databases of PubMed, Web of Science, Embase, and Google Scholar (as a search engine); only full-text available articles were considered, and articles published from 2008 to 2018 were prioritized. The search strategy included the combination of following keywords: "Helicobacter pylori", "anti-Helicobacter", "medicinal plant", "plant extract", "essential oil", "bioactive", "phytochemical", "antimicrobial", and "eradication".

Helicobacter pylori and Gastric Cancer
H. pylori infection has been implicated in the development of gastric cancer, a multifactorial disease and a leading cause of mortality. The risk factors for gastric cancer have been shown to include environmental factors and factors that influence host-pathogen interaction, as well as the complex interplay between these factors [31]. Modern lifestyle, high stress levels, smoking and excessive alcohol consumption, nutritional deficiencies, and prolonged use of non-steroidal anti-inflammatory drugs (NSAIDs) are amongst the most relevant etiological environmental factors [32].
This bacterial infection has been linked to the initiation of chronic gastritis that could later lead to adenocarcinoma of the intestine [33]. However, several mechanisms have been proposed to represent the involvement of H. pylori infection in tumorigenesis. Several bacterial virulence factors, such as the cytotoxin-associated gene A (CagA) protein, present in the DNA insertion element Cag pathogenicity island (CagPAI), were found to be of prominent importance in carcinogenesis [34]. Likewise, bacterial peptidoglycan can be delivered into gastric epithelial cells, where it activates a phosphoinositide 3-kinase (PI3K)-Akt pathway leading to cell proliferation, migration, and prevention of apoptosis [35]. Furthermore, H. pylori-induced gastric inflammation involves the cyclooxygenase-2 (COX2)/prostaglandin E2 (PGE2) pathway and inflammatory marker interleukin 1β (IL-1β), which are important factors triggering chronic active gastritis and The susceptibility of H. pylori isolates and strains to 543 extracts from 246 plant species was tested by disc diffusion, agar diffusion, agar dilution, and broth microdilution assays. Activity ranged from 1.56-100,000 µg/mL for minimal inhibitory concentration (MIC) and 7-42 mm for inhibition zone diameters (IZDs). However, disparities were observed among the methods used and the tested concentrations: some extracts were tested at very high concentrations (100,000 µg/mL) that might have resulted in biased conclusions. Though many plants (246 species) showed anti-H. pylori activity in vitro, very few have been screened for activity in animal models.
Organic extracts of Carum carvi, Xanthium brasilicum, and Trachyspermum copticum have demonstrated antibacterial activity against 10 clinical isolates of H. pylori [46]. In addition, ethanolic extracts of Cuminum cyminum and propolis exhibited significant in vitro inhibitory effect against H. pylori and, therefore, could be considered a valuable support in the treatment of infection, even contributing to the development of new and safer agents for inclusion in anti-H. pylori therapy regimens [47]. Some popular plant species used in Brazilian cuisine and folk medicine in the treatment of gastrointestinal disorders were also investigated for their antibacterial effects, among which Bixa orellana, Chamomilla recutita, Ilex paraguariensis, and Malva sylvestris were the most effective against H. pylori [48].

In Vivo Findings
H. pylori colonization is increasingly being associated with a heightened risk of developing upper gastrointestinal tract diseases. Despite many plant extracts having demonstrated a prominent H. pylori inhibition capacity in culture, it is of crucial importance to assess their in vivo efficacy, because it is pivotal to ascertain their effective antibacterial potency. However, a relatively low number of medicinal plants have been investigated to date for in vivo activity, as discussed below.
Paeonia lactiflora root extract (100 µg/mL) showed a complete inhibition of H. pylori colonization (4-5 × 10 5 colony forming unit (CFU)), being the antibacterial potential equivalent to of ampicillin used as positive control (10 µg/mL) (2-4 × 10 5 CFU) [98]. Time course viability experiments were also performed in simulated gastric environments to assess the anti-H. pylori activity of garlic (Allium sativum) oil (16 and 32 µg/mL). A rapid anti-H. pylori action in artificial gastric juice was found. Nevertheless, the anti-H. pylori activity displayed by garlic oil was noticeably affected by food materials and mucin, despite the fact that a substantial activity remained under simulated gastric conditions [65]. Also, H. pylori-inoculated Swiss mice receiving 125, 250, or 500 mg/kg of Bryophyllum pinnutum or ciprofloxacin (500 mg/kg) for 7 days, showed a significant reduction of H. pylori colonization on gastric tissue from 100% to 17%. In addition, the highest B. pinnatum extract tested (85.91 ± 52.91 CFU) and standard drug ciprofloxacin (25.74 ± 16.15 CFU) also reduced significantly (p < 0.05) the bacterial load of gastric mucosa as compared with untreated infected mice (11883 ± 1831 CFU) [74]. On the other hand, Eryngium foetidum methanol extract (381.9 ± 239.5 CFU) and positive control ciprofloxacin (248 ± 153.2 CFU) significantly reduced the bacterial load in gastric mucosa at the same dose (500 mg/kg) compared with untreated and inoculated mice (14350 ± 690 CFU) [73].
Hippocratea celastroides hydroethanolic root-bark extract, a widely used plant against gastric and intestinal infections, also showed anti-H. pylori efficacy in naturally infected dogs. In a study of 18 experimental dogs treated with a dose of 93.5-500 mg/kg of H. celastroides extract in weight and 19 infected dogs receiving amoxicillin-clarithromycin-omeprazole (control treatment), the results showed effectiveness of 33.3 and 55% in the experimental and control groups, respectively [99].
On the other hand, Ye et al. [95], aiming to investigate the in vivo bactericidal effects of Chenopodium ambrosioides L. against H. pylori, randomly assigned H. pylori-infected mice into plant extract group, triple therapy control (lansoprazole, metronidazole, and clarithromycin), blank control, and H. pylori control groups. The obtained eradication ratios, determined by rapid urease tests (RUTs) and histopathology, were, respectively, 60% (6/10) using RUT and 50% (5/10) using histopathology for the test group and both 70% (7/10) for the control group. In addition, the histopathologic evaluation revealed a massive bacterial colonization on the gastric mucosa surface and slight mononuclear cells infiltration after H. pylori inoculation, but no obvious inflammation or other pathologic changes in gastric mucosa were stated between the C. ambrosioides-treated mice and the standard therapy.
Tinospora sagittata and its main component, palmatine, showed in vitro bactericidal effects on H pylori strains, with both MIC and minimal bactericidal concentration (MBC) values of 6250 µg/mL, whereas palmatine's MIC value against H. pylori SCYA201401 was 6.25 µg/mL and against H. pylori SS1 was 3.12 µg/mL. The time-kill kinetic study evidenced a dose-dependent and progressive decline in the numbers of viable bacteria up to 40 h. H. pylori-infected mice treated with extract, palmatine, or control therapy (omeprazole, clarithromycin, and amoxicillin), presented eradication ratios of, respectively, 80%, 50%, and 70%. The anti-H. pylori activity found in T. sagittata extracts and its major constituent, palmatine, both in culture and animal models, clearly highlights the antibacterial potential of this plant in the treatment of both infected humans and animals [42].
Total alkaloids fraction activity (TASA) of Sophora alopecuroides L., widely used in herbal remedies against stomach-associated diseases, were also investigated on 120 H. pylori-infected BALB/c mice mouse gastritis. A total of 100 infected mice were randomly assigned into 10 treatment groups: group I (normal saline); group II (bismuth pectin); group III (omeprazole); group IV (TASA 2 mg/day); group V (TASA 4 mg/day); group VI (TASA 5 mg/day); group VII (TASA + bismuth pectin); group VIII (TASA + omeprazole); group IX (bismuth pectin + clarithromycin + metronidazole); and group X (omeprazole + clarithromycin + metronidazole). The mice were sacrificed 4 weeks after treatment. Real-time PCR was used to detect 16sDNA of H. pylori to test both the colonization and mice clearance of bacteria of each treatment. Hematoxylin and eosin staining and immunostaining of mice gastric mucosa were also used to observe the general inflammation and related factors: IL-8, COX2, and nuclear factor-kappa B (NF-κB) expression changed after treatments. TASA combined with omeprazole or bismuth pectin showed promising antimicrobial activity against H. pylori, as well as conventional triple therapy. Indeed, hematoxylin and eosin staining and immune-staining of mice gastric mucosa evidenced that the inflammation on mice gastric mucosal membrane were also clearly relieved in TASA combined treatments and conventional triple therapy compared with normal saline-treated mice. Accordingly, from immunohistochemistry results, H. pylori-induced IL-8, COX2, and NF-κB were consistently suppressed in the seventh, eighth, ninth, and tenth groups to a certain extent [100].
Pastene et al. [101] investigated the inhibitory effects of a standardized apple peel polyphenol-rich extract (Malus pumila Mill., cited as Malus domestica) against H. pylori infection and vacuolating bacterial toxin (VacA)-induced vacuolation and found that the preparation significantly prevented vacuolation in HeLa cells with an IC 50 value of 390 µg gallic acid equivalents (GAE)/mL and an in vitro anti-adhesive effect against H. pylori. A significant inhibition was also stated with 20-60% reduction of H. pylori attachment at concentrations between 0.250 and 5 mg GAE/mL. In a short-term infection model (C57BL6/J mice), doses of 150 and 300 mg/kg/day showed an inhibitory effect on H. pylori attachment. Orally administered apple peel polyphenols also showed an anti-inflammatory effect on H. pylori-associated gastritis, lowering malondialdehyde levels and gastritis scores.
Kim et al. [102] investigated the GutGard™ ability (a flavonoid rich, Glycyrrhiza glabra root extract) to inhibit H. pylori growth both in Mongolian gerbils and C57BL/6 mouse models. Infected male Mongolian gerbils were orally treated once daily 6 times/week for 8 weeks with 15, 30, and 60 mg/kg GutGard™. Bacterial identification in the biopsy samples of gastric mucosa, via urease, catalase, and ELISA, as well as immunohistochemistry revealed a dose-dependent inhibition of H. pylori colonization in gastric mucosa by GutGard™. As well, the administration of 25 mg/kg GutGard™ in H. pylori-infected C57BL/6 mice significantly reduced H. pylori colonization in gastric mucosa, suggesting its usefulness in H. pylori infection prevention.
Calophyllum brasiliense stem bark preparations are popular remedies for the treatment of chronic ulcers. A current report evidenced gastroprotective, gastric acid inhibitory properties and anti-H. pylori activity in culture (MIC = 31 µg/mL) [75]. Hydroethanolic (50, 100, and 200 mg/kg) and dichloromethane (100 and 200 mg/kg) fractions-treated Wistar rats ulcerated by acetic acid and inoculated with H. pylori, showed a marked delay in ulcer healing and reduced the ulcerated area in a dose-dependent manner [75]. While the dichloromethane fraction, at 200 mg/kg, increased PGE2 levels, both the hydroethanolic and dichloromethane fractions decreased the number of urease-positive animals, as confirmed by the reduction of the H. pylori presence in histopathological analysis. This aspect suggests that the antiulcer activity of C. brasiliense is partly linked with its anti-H. pylori efficacy [75]. Also, phenolic-rich oregano (Origanum vulgare) and cranberry (Vaccinium macrocarpon) extracts showed a prominent ability to inhibit H. pylori through urease inhibition and disruption of energy production by inhibition of proline dehydrogenase at the plasma membrane [103].

Urease Inhibition
The current therapies are challenged by the considerable number of emerging H. pylori-resistant strains. This fact has driven the need for alternative anti-H. pylori therapies that ideally should have good stability and low toxicity and to be able to inhibit urease activity [62]. It has been shown that H. pylori urease activity is crucial in bacterial survival and pathogenesis [104].
The inhibitory potency of some anti-H. pylori medicinal plants has been reported [62] and even investigated by some authors in the involved mechanisms of antibacterial action of those plant products [63]. Table 11 briefly shows the studied plant extracts with prominent anti-urease activity. Amin et al. [49] demonstrated that the methanolic and acetone extracts of some medicinal plants were able to inhibit urease activity. In fact, Acacia nilotica flower methanol and acetone extracts evidenced anti-H. pylori activity, being MIC values of 8-64 µg/mL and 4-64 µg/mL, respectively. Both extracts inhibited urease activity at 8.2-88.2% and 9.2-86.6%. Calotropis procera leaf and flower methanol and acetone extracts, with MIC values of 16-256 µg/mL, 32-256 µg/mL, and 8-128 µg/mL also displayed urease inhibitory effects, being, respectively, 12.2-48.2% and 7.2-58.2% for leaf and 9.3-68.2% for flower acetone extracts [49]. While A. nilotica extract exerted a competitive inhibition, that of C. procera extract displayed a mixed type of inhibition [49]. In addition, Casuarina equisetifolia fruit methanol extract, with MIC values varying from 128-512 µg/mL, also displayed 12.2-86.2% inhibition of urease activity [49].
In another study, Camellia sinensis young non-fermented and semi-fermented shoot extracts, presented inhibition zone diameter (IZD) and MBC of, respectively, 22.5 mm at 20-60 µg/disk and 4 mg/mL, and 18 mm at 20-60 µg/disk and 5.5 mg/mL. They both inhibited Ure A and Ure B subunits production at 2.5 and 3.5 mg/mL [94]. Also, the Chamomilla recutita flower extract, which inhibited H. pylori growth at an MIC 90 value of 125 mg/mL and a MIC 50 value of 62.5 mg/mL, was able to inhibit the urease production [105]. In the same line, the methanol fraction of Euphorbia umbellata bark extract inhibited both H. pylori growth (44.6% inhibition) at 256 µg/mL and urease activity (78.6% inhibition) at 1024 µg/mL [77]. Moreover, the Peumus boldus flower aqueous extract showed anti-adherent activity against H. pylori and inhibited urease activity with an IC 50 value of 23.4 µg GAE/mL [61]. The aqueous extract of Teminalia chebula fruit showed activity with MIC and MBC values of 125 mg/mL and 150 mg/mL, respectively, and inhibited H. pylori urease activity at a concentration of 1-2.5 mg/mL [63].

Conclusions and Future Perspectives
Overall, the report suggests that the studied plant extracts possess anti-H. pylori activity, strengthening the claims made by traditional medicine practitioners about their putative anti-ulcerative properties. However, very few of them were investigated for efficacy in animal models or the ability to inhibit urease activity. Further studies are warranted for efficacy studies in animal models, elucidation of effective modes of action (including urease inhibition), and clinical trials in human being.
Author Contributions: All authors contributed equally to this work. B.S., J.S.-R., P.V.T.F., and N.M. critically reviewed the manuscript. All the authors read and approved the final manuscript.
Funding: The APC was funded by N Martins.