Spectra of Antibacterial Activity of Propolis (Promax-C) Samples from Two Localities of Adamaoua Province (Cameroon)

INTRODUCTION

Since thousands of years, natural products have been used in folk medicine to treat several diseases. Among them, propolis has got an increased interest because of its antimicrobial activity spectra against a wide range of pathogenic micro-organisms (Sonmez et al., 2005). The word propolis is derived from the Greek pro which means for or in defense and polis for city, referring to a substance used to defend the city or the hive (Santos et al., 2002). Propolis is a complex resinous mixture collected by honeybees (Apis mellifera) from buds and exudates of certain plants. This resin is masticated, salivary enzymes are added and the partially digested material is mixed with wax and used by bees to seal cracks and crevices, smooth out the internal walls and protect the entrance of the hive against intruders (Molan, 2001; Sonmez et al., 2005). The chemical composition of propolis varies according to the plants that can be found in a specific region (Ghisalberti, 1979 ; Markham et al., 1996). More than three hundred constituents were identified in different propolis samples by Bankova et al. (2000). Flavonoids, aromatic acids, diterpenic acids and phenolic compounds appear to be the principal components responsible for the biological activities of propolis samples.

In general, propolis is used in a crude form or as ethanolic extracts. Many researchers reported the pharmacological properties of ethanolic extract of propolis such as antibacterial (Kujumgiev et al., 1999; Sforcin et al., 2000; Sorkun et al., 2001; Borelli et al., 2002; Kartal et al., 2003; Silici and Kutluca, 2005) antifungal (Kujumgiev et al., 1999; Ota et al., 2001; Sawaya et al., 2002; Kartal et al., 2003; Choi et al., 2006) antiviral (Manolova et al., 1985; Amoros et al., 1994; Gekker et al., 2005) anti-inflammatory (Miyataka et al., 1997), local anaesthetic effect (Paintz and Metzner, 1979), antioxidant (Volpert and Elstner, 1993; Orhan et al., 1999; Choi et al., 2006), immunostimulating (Dimov et al., 1991; Sforcin, 2007) and cytostatic effects (Banskota et al., 1998). Egyptians, Greeks and Romans used propolis to cure some lesions of the skin. In Cameroon, Promax-C is a new natural product prepared as ethanolic extract of propolis that is used by population to treat wounds, burns, respiratory and dental infections, stomach ulcer, etc.

The aim of the present study is to describe the antibacterial activity spectra of Promax-C samples, prepared from propolis collected in two localities of Adamaoua Province (Cameroon), in order to confirm the validity of their popular use as an antibiotic agent.

MATERIALS AND METHODS

This study was conducted from February 2006 to July 2007 in the Laboratory of Microbiology of the National Advanced School of Agro-Industrial Sciences, University of Ngaoundere (Cameroon) and the Laboratory of Science and Food Engineering of the National Polytechnic Institute of Lorraine (Nancy, France).

Characteristics of Promax-C Samples
Propolis origins and other properties of Promax-C samples analyzed are indicated in the Table 1.

Promax-C samples are propolis extracts in 70% (v/v) ethanol prepared and provided by AFH Association of Ngaoundere and kept in amber flasks. The 70% ethanol used for extraction of the Promax-C samples showed no bactericidal activity on bacteria tested.

Table 1:	Propolis origins and other properties of Promax-C samples analyzed

*Localities of Adamaoua Province (Cameroon)

Bacterial Strains
All the seven bacterial strains tested were provided by the Laboratory of Science and Food Engineering of ENSAIA-INPL (Nancy, France). They are:

•	Salmonella enterica sp. enterica CIP 81.3
•	Staphylococcus aureus CIP 7625
•	Escherichia coli CIP 54. 8
•	Enterococcus faecalis CIP 76117
•	Listeria monocytogenes CIP 82110
•	Pseudomonas fluorescens CIP 6913
•	Bacillus subtilis CIP 6624

Antibacterial Tests
Assay for Inhibition of Bacterial Growth
The well diffusion technique on agar medium was used to test the Promax-C samples against bacteria. To Petri dishes containing 15 mL of TSA-YE medium (Trypcase Soja Agar-Yeast Extract)+ tween 80 was added 0.15 mL of an 18 h pre-culture of the Bacillus subtilis strain or 0.015 mL of an 18 h pre-culture of others bacterial strains obtained in TSB-YE medium (Trypcase Soja Broth-Yeast Extract) and thoroughly mixed. After solidification of the medium, six wells of 6 mm diameter were created in each Petri dish and five of them loaded with 20 μL of different Promax-C samples. Twenty microliter of the solvent control (70% ethanol) were introduced in the remaining well per dish. Dishes were left in a refrigerator at 4°C for 24 h. The plates were incubated at 30°C for Pseudomonas fluorescens strain and 37°C for others bacterial strains during 18 h. After incubation, the diameter of the zone of growth inhibition (mm) around each well was measured. An inhibitory zone with diameter less than 6 mm corresponds to lack of activity of the sample. The solvent control (ethanol) did not show any antibacterial activity. All determinations were made in duplicate.

Determination of Minimal Inhibitory Concentrations (MICs)
The MICs were determined by the macrodilution method according to the National Committee of Clinical Laboratory Standard guidelines (Jorgensen et al., 1997). An 18 h pre-culture of the bacterial strains in a double concentration TSB-YE medium corresponding to an inoculum of approximately 10⁴ cfu mL^-1 (0.5 Mc Farlands) was prepared. Serial concentrations of Promax-C from different samples ranging from 0.5 to 14% (v/v) were achieved in test tubes with sterile distilled water and/or 70% ethanol to yield a total volume of 2 mL per tube. Each antibacterial assay also included tubes containing the culture medium inoculated or not and/or ethanol, in order to obtain controls of the solvents antibacterial effect. The test tubes were incubated at 30°C for Pseudomonas fluorescens strain and 37°C for others bacterial strains during 24 h. After incubation, plates were inoculated with 50 μL of each tube by a multipoint inoculator and incubated at 30°C for Pseudomonas fluorescens and 37°C for others strains during 24 h. The MIC endpoints were read as the lowest concentration of Promax-C that resulted in no visible growth on the surface of the culture medium. All tests were made in duplicate.

HPLC Analysis
The phenolic compounds of Promax-C samples were analyzed in a chromatograph (SHIMADZU 10A) equipment. The chromatographic conditions were reverse phase column (LichroChart PUROSPHER RP-18; 25.0x0.4 cm, particle diameter of 5 μm (Merck)). The mobile phase was water (solvent A) and methanol (solvent B), at a flow rate of 1 mL min^-1 at 30°C using a linear gradient, starting with 30% B (0-15 min) and increasing to 90% B (15-75 min), held at 90% B (75-95 min) and decreasing to 30% B (95-105 min). The time of analysis was 50 min and the detection was done with a diode array detector (SHIMADZU SPD-M10). Chromatograms were recorded at 268 nm for phenolic compounds quantification (Markham et al., 1996).

RESULTS AND DISCUSSION

All the Promax-C samples were active against all the Gram positive bacteria tested except E. faecalis (Table 2). On the contrary, there was no activity of the same propolis samples against all the Gram negative bacteria studied. The most susceptible bacteria strain to the Promax-C samples was L. monocytogenes for which was recorded the greatest inhibitory zone (5.8±0.1 mm) due to the most active sample namely Promax-C8. The most active propolis sample against S. aureus and B. subtilis was also the Promax-C8 with inhibitory zones of 5.6±0.2 mm and 4.6±0.3 mm, respectively. The susceptibility of the bacterial strain against the Promax-C samples tested decrease in the following order L. monocytogenes>S. aureus>B. subtilis.

Results of the susceptibility of the Gram positive bacteria (except E. faecalis) to the most active Promax-C samples are represented in Table 3 and show that the most susceptible strain to the Promax-C samples was L. monocytogenes and the least susceptible was B. subtilis. The most active propolis sample against two of the three most susceptible bacteria strains tested was Promax-C8 with a MIC<1% (v/v) for L. monocytogenes while the least active sample was Promax-C2 against B. subtilis with a MIC equal to 9% (v/v).

Considering the MIC values, the susceptibility of bacteria strains to the Promax-C samples decreased in the following order L. monocytogenes>S. aureus>B. subtilis and confirmed the results of the qualitative tests.

Results of HPLC analysis of phenolic compounds of the least active and the most active Promax-C samples are shown in Fig. 1a-f and Table 4 . These results showed that areas of the minor peaks and major peaks of phenolic compounds were less important for the least active Promax-C samples (Promax-C1, Promax-C2) than those of the most active Promax-C samples (Promax-C7, Promax-C8 and Promax-C13).

The well diffusion method on agar medium has been used to determine the inhibitory zones of four Gram positive bacteria strains and three Gram negative bacteria strains due to the activity of different Promax-C samples. All the Promax-C samples studied showed an activity against S. aureus, L. monocytogenes, B. subtilis except E. faecalis concerning the Gram positive bacterial strains. These results are in agreement with those of Choi et al. (2006), who showed an antibacterial activity of propolis against S. aureus and B. subtilis. On the contrary, the same Promax-C samples showed no activity against the Gram negative bacterial strains studied namely E. coli, S. enterica and Ps. fluorescens.

Table 2:	Antibacterial activity of Promax-C samples*

Diameter of the inhibitory zone±SD (mm); SD: Standard deviation; S.a: Staphylococcus aureus; S.e: Salmonella enterica; E.c: Escherichia coli; E.f: Enterococcus faecalis; L.m: Listeria monocytogenes; Ps. f: Pseudomonas fluorescens; B.s: Bacillus subtilis; -: No inhibition; *Mean values of two measurements

Table 3:	Susceptibility of the Gram positive bacteria (except E. faecalis) to the most active Promax-C samples

nd: Not determined

Table 4 :	Minor and major peaks area of phenolic compounds of the least active and the most active Promax-C samples obtained by HPLC

10x and 20x: Dilution rate; RT: Retention time in min; λ: Wavelength in nm

The E. coli resistance to ethanolic extracts of propolis was described by Drago et al. (2000) and Popova et al. (2005). Similar results were obtained by Grange and Davey (1990), Keskin et al. (2001), Ugur and Arslan (2004) and Silici and Kutluca (2005), who showed that propolis was more active against Gram positive bacterial strains than Gram negative strains.

Kujumgiev et al. (1993) and Greenaway et al. (1998) showed that fatty acid esters, phenolic compounds and cinnamic acid were the main propolis constituents and that some of them had an antibacterial activity. Silici and Kutluca (2005) had attributed the greater activity of the Apis mellifera caucasica propolis samples to its varied chemical composition and concentrations of constituents. The mechanism of antimicrobial activity of propolis is complex and could be attributed to a synergism between phenolic and other compounds in the resin (Kedzia, 1990; Krol et al., 1993). Popova et al. (2005) studied the qualitative and quantitative chemical composition of Turkish propolis and confirmed the importance of its phenolic compounds contents for the different antibacterial activity expressions. These researchers showed that the greater antibacterial activity of propolis samples from Central and Western Anatolia was linked to their high phenolic and flavonoid contents and that the lower activity of other samples against S. aureus was related to their low concentrations in these substances. Promax-C8 was the most active sample and its higher phenolic compounds concentrations could explain its greater activity.

S. aureus is a bacterial strain resistant to penicillin-G (Moreno et al., 1999). It is interesting to remark that the most active Promax-C samples could be used to treat skin affections due to that bacteria strain.

Promax-C samples studied showed an activity against all the Gram positive bacterial strain tested except E. faecalis. On the contrary, the same Promax-C samples showed no activity against the Gram negative bacterial strain tested. The more susceptible bacterial strain to the majority of Promax-C samples was L. monocytogenes while the less susceptible bacterial strain was B. subtilis. The most active sample namely Promax-C8 had the highest phenolic compounds content while the less active propolis sample, Promax-C1, had the lowest phenolic compounds amount. These findings showed that there is a relationship between the Promax-C phenolic compounds content and their antibacterial activity.




Fig. 1:	HPLC chromatograms of phenolic compounds of the Promax-C samples that exhibited weak or high antibacterial activity. (a) Promax-C8, (b) Promax-C7, (c) Promax-C13, (d) Promax-C6, (e) Promax-C2 and (f) Promax-C1

The notable antibacterial activity of the most active Promax-C samples obtained in present results could justify their use in the treatment of affections due to some of the bacterial strain tested.

ACKNOWLEDGMENTS

We are grateful to Prof. Jean-Bernard Milliere and his collaborators in the Laboratory of Science and Food Engineering of ENSAIA-INPL (Nancy, France) for their contribution in the realization of this study.