The discovery of Helicobacter pylori (H. pylori) in 1982 was the starting point of a conceptual revolution concerning of gastroduodenal diseases and their management [1]. H. pylori infection has been implicated in the pathogenesis of chronic gastritis, peptic ulcers and, more rarely, gastric cancer and gastric lymphoma of mucosa-associated lymphoid tissue [2]. The mechanisms by which bacterial infection leads to gastric mucosal damage include the direct effects of virulent factors produced by H. pylori, the propagation and perpetuation of inflammation, oxidative stress, and the induction of apoptosis in infected gastric epithelial cells [3].

Under inflammatory conditions, phagocytosing cells generate multiple well-defined reactive oxygen species (ROS). Stimulated polymorphonuclear neutrophils (PMNs) undergo an oxidative burst, and release large quantities of superoxide anion as a result of the activation of the PMN NADPH oxidase. Superoxide anion radicals are known to dismutate to form hydrogen peroxide (H₂O₂) and oxygen [4]. Myeloperoxidase (MPO), an enzyme released from the azurophilic granules in neutrophils, uses H₂O₂ and chloride ions (Cl⁻) as substrates to produce hypochlorous acid (HOCl), an important antibacterial compound, but an extremely strong oxidant that can also attacks host biomolecules [5]. Oxidative stress plays a critical role in the augmented mucosal damage caused by H. pylori infection, and an antioxidant could ameliorate the aggravation caused by stress-associated gastric mucosal damage.

Despite years of experience with H. pylori treatment, an ideal regimen to treat the infection has not yet been identified. The most effective treatment is a combination of a proton pump inhibitor and antibiotics, but this fails to eradicate the infection in 10–20% of patients [6]. Non-antibiotic treatments, including phytomedicines, probiotics and antioxidants, have been increasingly investigated as potential alternatives for the treatment of H. pylori infection [7,8].

Byrsonima crassa (Malpighiaceae) is a plant found in the Cerrado of the central region of Brazil and is used in folk medicine for the treatment of gastroduodenal diseases, including gastric ulcers. Pharmacological studies have revealed that Byrsonima species have an antiulcerogenic effect and that the methanolic extract of leaves from B. crassa has a gastroprotective effect against HCl/ethanol-induced gastric mucosal injuries in mice. Amentoflavone, catechin and quercetin derivatives have been suggested to be the active components of the extract [9].

Considering that B. crassa is commonly used as a phytomedicine to treat ulcers and gastritis, the objective of the present study was to evaluate the effect of the extract and its purified major phenolic constituents (amentoflavone, (+)-catechin, methyl gallate, quercetin 3-O-α-l-arabinopyranoside, and quercetin 3-O-β-d-galactopyranoside) on the oxidative burst of PMNs stimulated by H. pylori, using a luminol-dependent chemiluminescence assay, and the anti-H. pylori activity of each compound.

There is considerable interest in alternative approaches to the treatment of H. pylori, such as the use of biologically active compounds, including antimicrobials compounds and antioxidants from plants. In a previous study, the anti-ulcerogenic activity profile of B. crassa leaves was investigated in detail, in various in vivo experimental ulcer models [9]. In this study the effects of the whole extract and purified phenolic compounds on the intra- and extracellular production of ROS was assessed in H. pylori-stimulated PMNs using luminol-enhanced chemiluminescence. Luminol can be oxidized by several ROS, but it is generally accepted that chemiluminescence in neutrophils results from intra- and extracellular events and depends mainly on the reactions of the MPO-H₂O₂-Cl system [10]. The suppressive activity on oxidative burst of H. pylori-stimulated PMNs was in the order of methyl gallate > (+)-catechin > methanol extract > quercetin 3-O-α-l-arabinopyranoside > quercetin 3-O-β-d-galactopyranoside > amentoflavone. All compounds showed a dose-dependent effect (Table 1).

In general, the free-radical scavenging and antioxidant activities of phenolics depend primarily on the number and positions of the hydrogen-donating hydroxyl groups on the aromatic ring of these molecules, but is also affected by other factors, such as glycosylation of aglycones and other H-donating groups [11]. The experimental results of this study showed that the flavonoid quercetin (standard antioxidant) had a better antioxidant activity than its 3-O-glycoside derivatives (quercetin 3-O-α-l-arabinopyranoside and quercetin 3-O-β-d-galactopyranoside) on ROS production induced by H. pylori. These agree with reports that flavonoid aglycones are more potent antioxidants than their corresponding glycosides [12].

Methyl gallate has been shown to be an effective antioxidant in a variety of acellular experiments [13]. In this study, this gallic acid derivative showed a strong inhibitory activity on the induced oxidative stress of PMNs using H. pylori as activator. The molecular mechanism for ROS production by H. pylori remains unclear. Analysis of intracellular ROS shows that methyl gallate is effective in attenuating H₂O₂-derived ROS [14]. The antioxidant-like properties of polyphenols are largely dependent on the type of stimulus for the production of ROS and the structure plays a critical role in the success as an antioxidant. The results of ROS inhibitory activity of different phenolic compounds are indicative of different action mechanisms and further researches are required to understanding their action on ROS induced by H. pylori in neutrophils. One of the initial components of the innate immune response to be encountered by H. pylori in the stomach is the gastric epithelial cell [3]. As B. crassa and phenolic constituents have impact on ROS induced by PMNs is expect that this benefits can also be extent to the gastric mucosal cells.

In this study the anti-H. pylori activity of the phenolic compounds isolated from the methanolic extract of B. crassa was also examined (Table 2). All purified compounds tested showed lower anti-H. pylori activity than did the whole extract as cited by Bonacorsi et al. [15]. Studies have demonstrated the anti-H. pylori effect of natural compounds [16,17]. These compounds interact with multiple molecular targets and inhibit the growth of H. pylori by various mechanisms such, as membrane destabilization, inhibition of ion channels and inhibition of bacterial metabolism [14]. Phenolic compounds isolated from B. crassa weakly inhibited H. pylori growth. Shin et al. [18] have previously reported that some flavonoids, such catechins, quercetin, and naringenin, exhibit poor inhibition on H. pylori growth.

Synergistic antimicrobial activity has been demonstrated in some naturally occurring flavonoids. Arima et al. [19] reported that the use of combinations of quercetin and quercitrin, quercetin and morin, and quercetin and rutin were more effective against S. enteritidis than the use of each flavonoid alone. In a study of the effects of cranberry fruit on H. pylori, Vattem et al. [20] report the low efficacy of purified phenolics in inhibiting the bacteria compared to the whole fruit extract at a similar dose, suggesting the ability of phenolics to function synergistically in the whole food. The purified phenolic compounds tested showed a lower antimicrobial activity compared to the extract (MIC 1024 μg/mL) [15]. This result might reflect the synergistic interaction of constituent phytochemicals.

The present investigation constitutes the first quantitative screening for the effects phenolic compounds on the oxidative burst of PMNs induced by H. pylori. It becomes clear that B. crassa may exert a protective effect by inhibiting the mechanism by which H. pylori and neutrophils collaborate to cause gastric mucosal damage. These results confirm the antioxidant activity of B. crassa that justify its use in non-conventional medicine by the Brazilian population for the treatment of gastroduodenal ulcers, particularly when H. pylori displays its evasion mechanism.