Inhibition of Helicobacter pylori growth in vitro by Bulgarian propolis: preliminary report

Propolis is a resinous product of bees with anti-inflammatory, antimicrobial, anaesthetic and immunostimulating activities (Koo et al., 2000). There are few data concerning the antibacterial activity of bee glue, mainly Brazilian propolis, on Helicobacter pylori (Banskota et al., 2001). However, several studies have reported that propolis has only a limited inhibitory effect against Gram-negative bacilli (Drago et al., 2000), and the chemical composition of bee glue exhibits geographical differences. There are no data about the effect of bee glue on Campylobacter spp. The aim of the study was to assess the inhibitory effect of Bulgarian propolis on the growth of 38 H. pylori and 18 Campylobacter clinical isolates in vitro by two methods. Thirty-eight H. pylori and 18 Campylobacter strains (Campylobacter jejuni, n = 10; Campylobacter coli, n = 8) were included in the study. Stock cultures were maintained in 15 % glycerol broth at -70 °C. They were subcultured onto MuellerHinton agar with 5 % sheep blood and 1 % IsoVitaleX (BBL), and incubated microaerobically at 35 °C for 4872 h. Inocula, corresponding to a value of 2 on the McFarland optical density scale, were prepared in MuellerHinton broth and were plated onto MuellerHinton agar plates in three directions by sterile swabs. The plates were left to dry for 15 min.

Activity of ethanolic extract of Bulgarian propolis (30 % EEP, w/v, Hygitest) was tested against 38 H. pylori strains by an agar-well diffusion method (AWDM). Ethanol (96 %) was used as a control. Wells, 7 mm in diameter, were punched in each agar plate using a sterile stainless steel borer. Each well was filled with 30, 60 or 90 µl 30 % EEP or 30 µl 96 % ethanol. In addition, a disc diffusion method (DDM), using paper discs (6 mm in diameter) containing either 5 µl 30 % EEP or 5 µl 96 % ethanol, was performed for 26 H. pylori and 18 Campylobacter strains. Two kinds of disc were used: moist propolis discs were prepared immediately before testing, and dry propolis discs were prepared in the same way and left to dry for 23 days.

The plates, tested by both methods, were incubated microaerobically (Helico-Campy Pack gas-generating envelopes, NCIPD, Bulgaria) at 35 °C for 72 h. The diameters of inhibitory zones were measured in mm. All isolates were tested in duplicate and mean values of growth inhibition for each strain were taken into account. The χ² test with Yates correction was used as a statistical method to determine significance.

Mean diameters of H. pylori growth inhibition by Bulgarian propolis are presented in Table 1. At a volume of 30 µl per well, 30 % EEP exhibited greater activity against H. pylori than did ethanol (mean diameters of growth inhibition: 17.8 vs 8.5 mm, P < 0.01). At volumes of 90 µl EEP per well, around 90 % of H. pylori strains exhibited large diameters of growth inhibition ( 15 mm, as shown in Fig. 1), vs 52.6 % by 30 µl EEP per well (P < 0.01). The results obtained by DDM were similar. The activity of moist propolis discs against H. pylori was slightly greater than that against Campylobacter isolates (mean inhibitory diameters: 21.4 vs 13.6 mm). Seventeen of 26 H. pylori isolates (65.4%) showed considerable growth inhibition ( 15 mm), vs 33 % (6/18) of Campylobacter strains (P>0.05). Ethanol exhibited a slight inhibitory effect on H. pylori, with inhibitory zone diameters of at least 15 mm in only 23.1 % of isolates. Although less active than the moist EEP discs, propolis in dried discs retained a residual antibacterial activity, inducing considerable growth inhibition ( 15 mm) in 34.6 % (9/26) of H. pylori strains and in 11.1 % (2/18) of Campylobacter isolates.

Table 1. Antibacterial activity of 30 % EEP and 96 % ethanol against H. pylori strains, as measured by AWDM and DDM

(127K): Fig. 1. Inhibitory effect of ethanolic extract of Bulgarian propolis (30 %) on H. pylori growth by AWDM. Note the large inhibitory zone around the well containing 90 µl propolis extract.

In different propolis samples, various substance combinations are responsible for the antibacterial activity of the bee glue. In Bulgaria and several Mediterranean countries, propolis contains mainly flavonoids and esters of caffeic and ferulic acids (Velikova et al., 2000). In propolis samples from the temperate zone, flavonoids and esters of phenolic acids are known to be associated with antibacterial activity (Kujumgiev et al., 1999). Although the inhibitory effect of propolis on Gram-positive bacteria has been demonstrated, the activity of bee glue against Gram-negative bacteria is a matter of controversy (Drago et al., 2000); for example, propolis has shown good activity against Haemophilus influenzae and Moraxella catarrhalis, but not against Enterobacteriaceae. The anti-H. pylori activity of Brazilian propolis has recently been reported, labdane-type diterpenes and some prenylated phenolic compounds being the main antibacterial substances (Banskota et al., 2001).

In the present study, the agar-well diffusion and the disc diffusion methods were used because they have the advantage of showing both inhibition and control growth (outside the inhibitory zone) of fastidious organisms on the same plate. Bulgarian propolis has considerable antibacterial activity against H. pylori in vitro: only 21 % (8/38) of the strains exhibited no inhibitory zone by AWDM using 30 µl EEP per well, and all isolates were inhibited to some extent by 90 µl EEP per well. Similar results were obtained by DDM. Only 3.8 % of H. pylori strains were not inhibited by moist EEP discs. Mirzoeva et al. (1997) have reported the species-dependent antibacterial effect of propolis, with some active, but labile, ingredients showing the highest activity. In the present study, the slight activity of dried propolis discs on H. pylori and Campylobacter strains, with mean inhibitory zone diameters of 12.4 and 11.6 mm, respectively, also suggests the presence of relatively stable antibacterial compounds.

In conclusion, the eradication of H. pylori infection is sometimes difficult because of increasing resistance to clarithromycin and metronidazole, the two major antimicrobial agents used in current triple regimens (Megraud, 2000). This motivates the search for alternative or additional therapeutic agents. The inhibitory activity of propolis against H. pylori in vitro is worth further bacteriological, pharmacological and clinical evaluation. The use of propolis mouthwashes could reduce or eliminate H. pylori in the mouth cavity, as a route of transmission of H. pylori infection (Megraud & Broutet, 2000). The synergistic effect of propolis and several antimicrobial agents (e.g. cloxacillin and doxycycline) has been demonstrated against Staphylococcus aureus (Krol et al., 1993). The presence or lack of synergistic effect of propolis with metronidazole, clarithromycin or amoxicillin against H. pylori is worthy of investigation. In addition, the anti-inflammatory and tissue-regenerative properties of propolis (Koo et al., 2000) could be an additional advantage in the prevention or treatment of H. pylori infection.