Volatile compounds in the stem bark of Sclerocarya birrea (Anacardiaceae) possess antimicrobial activity against drug-resistant strains of Helicobacter pylori

Abstract

The aim of this study was to isolate and identify phytochemicals with anti-Helicobacter pylori activity from the stem bark of Sclerocarya birrea. The plant crude extract was fractionated by silica gel column and thin layer chromatography techniques, initially with ethyl acetate (EA) and subsequently with a combination of ethyl acetate/methanol/water (EMW). Further fractionation and identification of the phytoconstituents was achieved by gas chromatography and mass spectrometry (GC/MS) analysis. The antimicrobial activity of the fractions and compounds was evaluated against five metronidazole- and clarithromycin-resistant strains of H. pylori as well as a reference strain ATCC 43526 using the microbroth dilution technique. Amoxicillin was included in the experiments as a positive control antibiotic. Of the 18 fractions collected, 16 demonstrated anti-H. pylori activity with 50% minimum inhibitory concentration (MIC₅₀) values ranging from 310 μg/mL to 2500 μg/mL. Two of the fractions (EMW fraction 6 and EA fraction 1) revealed the presence of 5 and 24 compounds, respectively, representing 40.5% and 86.57% of the total composition. Most of the compounds were essential oils, with terpinen-4-ol being the most abundant agent (35.83%), followed by pyrrolidine (32.15%), aromadendrene (13.63%) and α-gurjunene (8.77%). MIC₅₀ ranges for amoxicillin, terpinen-4-ol and pyrrolidine were 0.0003–0.06 μg/mL, 0.004–0.06 μg/mL and 0.005–6.3 μg/mL, respectively. The inhibitory activities of terpinen-4-ol and pyrrolidine were similar to amoxicillin (P > 0.05). Most of these compounds are being reported in this plant for the first time and may represent new sources of therapeutically useful compounds against H. pylori.

Introduction

Helicobacter pylori colonises the gastric mucosa of one-half of the world's population [1]. Infection may result in a number of gastroduodenal conditions, including peptic ulcer, gastric carcinoma and mucosa-associated lymphoid tissue (MALT) lymphoma [2], [3]. Use of combination therapy involving antibiotics with proton pump inhibitors or bismuth compounds has proven effective in the eradication of H. pylori infections [4]. However, combination therapy is plagued with undesirable side effects (e.g. epigastric pain, abdominal discomfort, diarrhoea, nausea and vomiting) [5]. As a result, patients often do not complete the treatment course, thus generating suboptimal antibiotic blood concentrations that predispose to the selection and survival of resistant bacterial strains [6]. Equally important is the fact that H. pylori colonisation of gastric epithelial cells and the mucus gel layer may result in the formation of protective biofilms reported to be more resistant to killing by antibiotics [7]. It therefore becomes imperative for combination therapy to be modified with more potent new agents capable of penetrating or dislodging these structures to enhance the efficacy of therapy.

Antibiotic resistance to commonly used drugs has become a major cause of treatment failure in the eradication of H. pylori infections, with failure rates of up to 40% [8]. These factors, as well as others including cost of combination therapy and the non-availability of some of the drugs in rural areas (especially in the developing world), have necessitated the search for new prophylactics and therapeutic alternatives [9].

The folkloric use and anecdotal evidence of some plants has shown great promise in the discovery of novel therapies against H. pylori infections [10], [11]. One such plant is Sclerocarya birrea, a popular medicinal plant amongst the Zulus, Vhavendas, Xhosas and Sothos of South Africa [12]. The leaves and stem bark of this plant are widely used in treating a plethora of stomach illnesses including gastritis, peptic ulcers and stomach cancer in South Africa and other African countries [13], [14].

Despite its numerous medicinal uses, the activity of this plant has rarely been evaluated against H. pylori, a risk factor and causative agent of gastritis, peptic ulcers and stomach cancer [2], [15]. An Internet search revealed a scarcity of information on the antibacterial activity of S. birrea and specifically on its anti-H.-pylori activity. There is a critical need for new therapies against H. pylori considering that susceptibility patterns are changing globally and current therapies are rendered obsolete by resistant bacterial strains. Interestingly, our previous studies demonstrated that crude acetone extracts of S. birrea are active against H. pylori [16]. It was therefore necessary to identify the active principle in these extracts as a continuation of the search for potent therapies against this notorious pathogen in a bid to circumvent the overall burden of antimicrobial resistance.