PROPOLIS: ANTIMICROBIAL ACTIVITY AND CHEMICAL COMPOSITION ANALYSIS

Over the last few years, propolis has been the object of many studies conducted around the world, and its biological properties and chemical composition have been widely investigated. The present study focuses on the evaluation of the antimicrobial activity as well as an examination of the chemical composition of two samples of propolis from Eastern Algeria coming from the commune of El Mechrouha and Ouled Driss in the wilaya of Souk-Ahras. The two samples are tested for their antimicrobial power by undertaking the agar diffusion technique on eight pathogenic microbial strains (six bacterial strains and two fungal strains) which are: Escherichia coli, Klebsiella pneumonia, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus thoraltensis, , , Candida famata and Aspergillus niger. The results obtained clearly show the impact of propolis on the microbial susceptibility of Gram-positive bacteria (S. agalactiae and S. aureus), as well as on fungal species (C. famata and A. niger). The analysis of the chemical composition of the ethanolic extracts of the two propolis by UV-visible absorption spectrometry and thin layer chromatography showed that Algerian propolis is wealthy in phenolic compounds, and high performance liquid chromatography allowed the identification of four polyphenols (Gallic acid, Caffeic acid, Quercetin and Catechin). These outcomes permitted a first assessment of the two propolis which present comparable components in their chemical compositions.

The extract obtained is called Ethanolic Propolis Extract (EPE: EPE1 Ethanolic Propolis Extract of El Mechrouha and EPE2 Ethanolic Propolis Extract of Ouled Driss). This extract is subjected to the study of the chemical profile and the determination of the antibiogram (Segueni, 2011).

Antibiogram by agar diffusion method (disc method)
The NCCLS technique (National Commite for Clinical Laboratory Standars) is used in this study.
The antibiogram is based on the observation of strain growth in the presence of a concentration gradient of an antimicrobial substance, obtained by diffusion from the discs into a Mueller Hinton (MH) agar medium (CA-SFM, 2010). For Streptococci, fresh blood MH was used.
-Place the discs in glass vials for sterilisation in an autoclave for 20 minutes at 120°C. -Using a Pasteur pipette or the sterile platinum handle, remove a microbial colony.
-Prepare the inoculum of each microbial strain and inoculate it onto the MH medium.
-Place each paper disc with sterile forceps on the MH medium and immediately impregnate it with 50μL with ethanolic extract of propolis 60%, 70%, 80% and 95%.
-Incubate the dishes at 35°C within 30 min after preparation and leave for 16 to 18h.
Accurately measure the diameters of the inhibition zones using a Caliper.

Extraction and purification of the chemical components of propolis
Flavonoic products are sought by UV-visible absorption and separated by thin layer chromatography (TLC) and high performance liquid chromatography (H.P.L.C).

UV-visible absorption
The concept is based on the absorption of light by the chemical species, the apparatus includes a white light source, a dispersive system that allows us to select the wavelength of the radiation and a detector system for measuring the light intensity of the monochromatic radiation that passes through the solution. The spectrophotometer compares the incident and transmitted light intensities by means of an electronic circuit that indicates the absorbance (Zeghad, 2009).
The UV-visible absorption spectra of EPE 1 and 2 are performed according to the method used by Park and Ikegaki (1998) (Segueni, 2011).
-Mix well and leave at room temperature for one hour.
We used a JENWAY UV 6705 spectrophotometer to record absorbances at wavelengths ranging from 200 to 600 nm. Measurements were carried out in quartz vats with an optical pathway of 1 cm (Kitouni, 2007).

Thin layer chromatography (TLC)
This technique is based on the partition of the different constituents of an extract according to their migration force in the mobile phase which is generally a mixture of solvents adapted to the type of separation sought and their affinity for the stationary phase which can be a polyamide gel or a silica gel. It allows us to have the fingerprints of the polyphenolic flavonoic content of the extract (Bobbitt et al., 1968). Preparation of the stationary phase: TLC was performed on pre-poured silica gel plates. Preparation of the mobile phase: The mobile phase consists of a mixture of organic solvents. For this purpose, different solvent systems have been tested to define those that give the best separations with different proportions as follows: 1. 95% ethanol / distilled water (V/V: 55/45), 2. n-butanol/acetic acid/distilled water (BAW) (V/V/V: 4/1/5), 3. Petroleum Ether/Ethyl Acetate (V/V: 7/3) (Zeghad, 2009).
-The deposition is done with disposable glass capillary tubes in a perpendicular and linear way. Plate development: Each plate is deposited in a vertical or slightly inclined position in the vat previously saturated by the vapors of the appropriate solvent system, the sample to be studied will be more or less entrained by progressive capillarity of the mobile phase towards the top of the plate (Zeghad, 2009). Revelation: If the constituents are colored, they will be directly visible on the plate, otherwise they can be revealed by UV light, which allows the UVabsorbing substances between 254 nm and 365 nm to be highlighted as spots.

Identification of flavonoids:
The behavior of a particular molecule in a given system is expressed by its fluorescence under UV light and by its Retention factor (Rf). Rf = distance between the origin and the spot of the product /distance from the origin to the solvent front

Structure-Rf
The migration distance of the substances depends mainly on their polarity as well as on their structures for example: The increase of (OH) causes a decrease in Rf, Methylation of (OH) groups and Acetylation leads to an increase in Rf values, whereas Glycosylation causes a decrease in Rf values mainly due to the introduction of new (OH) groups. Polyhydroxyflavones have low Rf values (0.00-0.25). Oligohydroxyflavones and oligomethoxyflavones have Rf values between (0.3-0.5). Flavanones, flavonols, methoxyflavones have the highest Rf values (0.5-0.75) (Zeghad, 2009).

Structure-fluorescence
Ultraviolet light examination is the most widely used method for the determination of the structure of flavonoids (Zeghad, 2009).

High Performance Liquid Chromatography (H.P.L.C.) Analysis
HPLC is a very powerful separation technique, it is widely used in many industries such as food, chemical and pharmaceutical industries, cosmetics, etc... It is a physico-chemical method based on the differences in interactions between the molecules to be separated and the mobile and stationary phases. Beforehand, the solutes are put in solution in the mobile phase (solvent). After its injection, this mixture passes under high pressure through the column (stainless steel tube) which contains the stationary phase (Nollet and Toldra, 2012). The analyses were performed using an HPLC-C18 chromatograph, furnished with the following elements: -A column (with a length of 125 mm and an internal diameter of 4.6 mm) containing the apolar stationary phase (reverse phase), the latter consisting of silica chemically modified by grafting residues (C-18), these reverse phase columns allow the separation of polar compounds, soluble in water or in hydroalcoholic mixtures; -A pumping system, Pump: Varian 9010, to move the mobile phase at high pressure (several tens of bars); -One injector: Varian 9100, to introduce the sample into the high-pressure system ; -A monochrome detector: Varian 9065; -Computer software to visualize the signals recorded by the detector. The working conditions are as follows: -Flow rate: 0.5 ml/min; -Working pressure: 100-150 bar; -Injection volume: 30 μl; -Wavelength: 254 nm; -Sample concentration: 1-5 mg/ml; -Analysis time: 15 min; The mobile phase is of constant composition, it is composed of a methanol-water mixture (60: 40 V/V) (Kuntić et al., 2007).

Results of the evaluation of the antimicrobial activity of propolis
We studied the antimicrobial potency in vitro of the ethanol extracts of the two propolis by the disc diffusion method on a solid agar medium, Mueller Hinton (MH). Discs impregnated with 50 μl of EPE1 and EPE2 with the 4 different concentrations of ethanol at 60%, 70%, 80% and 95% were tested by the NCCLS method. The antimicrobial activity of the extracts was estimated regarding the diameter of the inhibition zone around the discs containing the extracts to be tested against pathogenic microorganisms which are: S. aureus, S. agalactiae, S. thoraltensis, K. pneumonia, E. coli, P. aeruginosa, C. famata and A. niger. Ethanol has been tested as a solvent, the results show that it is suitable and has no effect on the normal growth of microbial strains. Results of the diameters of the inhibition zones show that 80% EPE gives the largest diameters for all strains. According to Table 1, the highest zones of inhibition were observed against A. niger (20 mm and 18 mm), S. aureus (16 mm and 15 mm) and S. agalactiae (14 mm and 12 mm), treated with EPE1 and EPE2 at 80%. Propolis from Ouled Driss (EPE2) scored a diameter of 10.5 mm against the yeast C. famata greater than that obtained with propolis from El Mechrouha (8.25 mm).  Legend: (-) -Absence of the inhibition zone, EPE (1) -Ethanolic Propolis Extract of El Mechrouha, EPE (2) -Ethanolic Propolis Extract from Ouled Driss

UV-visible absorption spectrum
The results of the UV-visible spectral analysis of the two propolis show a single absorption peak (Figure 1), which corresponds to band I (between 300 and 350 nm) of flavonoids. We assume that these peaks correspond to Flavones, Flavonols and Flavanones.

Thin layer chromatography (TLC)
Three solvent systems were used, the resulting chromatograms have a series of spots ( Figure 2). The chromatograms show that the number of spots as well as the colours obtained vary according to the solvent system used and the wavelength of the UV lamp (254-365 nm).
The identification of the components depends on the comparison of Rf and the colour observed under UV light (Tab 2 and Tab 3). These tables include the Rf of the different spots that appeared with the different solvent systems used as well as the colour revealed under UV light at two different wavelengths (254 and 365 nm). Using these solvent systems and at a wavelength of 254 nm, we were able to highlight: five spots with the solvent system (95% ethanol/distilled water), two spots with the solvent system (BAW) and seven spots with the system (petroleum ether and ethyl acetate), with colours that vary between brown-black, brown and violet; and an almost similar Rf for the two EPEs. On the other hand, the wavelength of 365 nm revealed: five spots with the first solvent system, four spots with the second and thirteen spots with the third solvent system, which show colour variability (blue, violet and yellow fluorescence); with almost similar Rf for the two EPEs. We noticed that the third solvent system (petroleum ether/ethyl acetate) gave the highest number of spots, revealed distinct spots and showed a considerable richness of flavonic substances in the two EPEs analysed, unlike the other two solvent systems which revealed a streak, which explains a poor separation of the components.

High Performance Liquid Chromatography (H.P.L.C.) Analysis
The results of the HPLC-C18 analysis of the active extract P1 are shown in Figure 3. As can be observed (depending on the number of peaks on the chromatograms), the extract is richer in chemical substances and this confirms the results obtained by TLC. Six pure phenolic compounds (Gallic Acid, Tannic Acid, Caffeic Acid, Catechin, Rutin, and Quercetin) were used in the H.P.L.C. analysis as controls. Their chromatograms and retention times (Rt) are shown in

Choice of solvent
We used ethanol (at different percentages) as a solvent for the study of antimicrobial activity for the following reasons: -Propolis cannot be utilized directly as a crude material because it is difficult to establish simple fractionation to get compounds because of its complex structure. The usual methodology is the utilization of a solvent, which should eliminate impurities and retain the desired components. Since the composition of propolis depends mainly on the vegetation from which it was gathered, but also on the methods utilized for extraction, the solvent used for the extraction of bioactive compounds should be carefully chosen (Fokt et al., 2010).
-A current and common procedure is to extract the alcohol-soluble fraction (Ghisalberti, 1979).
-Ethanol is the most common choice of solvent, as it allows the extraction of different classes of chemical components such as Polyphenols and Flavonols, and studies concerning the assessment of the bioactivity of propolis have been carried out using mainly ethanol extracts of propolis (Fokt et al., 2010).
-Ethanol is used in the composition of several therapeutic preparations (Brehon  et al., 2000). It evaporates easily and solubilizes the active components of propolis (Krell, 1996). Its effectiveness in the study of antimicrobial activity is confirmed by   et al., 1999). Chinese and Japanese propolis produces inhibition zones going from 5.5 to 6.8 mm with S. mutans (Ikeno et al., 1991). Stepanović et al. (2003) found that the propolis inhibition zone in different parts of Serbia was between 18 to 23 mm. Our findings are in the similar range as those revealed in this study, but the literature demonstrates that the susceptibility of microorganisms and differences in the active components of propolis that have antibacterial and antifungal activities are strongly influenced by changes in geographical origins (Bankova et al., 2000). The low susceptibility identified for E. coli was consistent with many publications, where it was inferred that this bacterium had a very low sensitivity to the bactericidal activity of propolis ( (Fokt et al., 2010). We suspect that this variation might be associated with the chemical composition of propolis. Bonvehi and Gutiérrez (2012) show that the antioxidant activity of Basque propolis varies with differences in phenolic compounds. Takaisi-Kikuni and Schilcher (1994) reveal by electron microscopy and micro-colorimetric analyses that EPE interferes with the division of S. agalactiae by pseudo-multicellular formation, disruption of the cytoplasm, inhibition of protein synthesis, causing bacterial lysis (Fokt et al., 2010). Mirzoeva et al. (1997) report that EPE and some phenolic components affect the bioenergetic state of the membrane by inhibiting the membrane potential, causing enhanced membrane permeability to ions and immobility of B. subtilis. In general, the two EPEs tested inhibit the strains studied and result in diameters that vary depending on the origin of the propolis, the species considered and the percentage of alcohol used.

Extraction and purification of active components
Flavonoids can be considered as pigments that absorb UV radiation very strongly, consequently UV-Visible spectroscopy represents the principal method for the structural examination of flavonoids. Flavones and flavonols are characterized in majority by two major absorption bands ( We also noted that EPE1 and EPE2 appear to have compounds in common with similar profiles, but with different concentrations depending on the intensity of the tasks obtained. The presence of flavones and flavonols has been observed by Bankova et al. (1982) in different propolis from the tropical region (Segueni, 2011). Segueni (2011) obtained between 4 and 5 spots using the solvent system 95% ethanol/distilled water. Propolis is an active product of the hive, it represents a remarkable chemical polymorphism. In the same extract, the biochemical content is very varied. Our results affirmed that the analysed samples had a high antimicrobial effect and flavonoid components are the most appropriate contenders for the assessment of the Algerian propolis quality, because of their diverse biological characteristics and their predominance in the phenolic fraction. Likewise, the higher the content of phenolic and flavonoid compounds, the greater the antimicrobial effect identified in the analysed extracts. H.P.L.C. was not performed for the second propolis sample (EPE2) due to lack of product. The results of the HPLC analysis are in accordance with those of Barrientos et al.

CONCLUSION
Natural substances occupy a large percentage of our daily life and especially in its large therapeutic choice. Indeed, bee propolis constitutes a real chemical bank from which we must take maximum advantage for the well-being of human beings. Our work, which is devoted to the study of the antimicrobial property of two samples of propolis from Eastern Algeria, has enabled us to observe that the field of beehive products is a vast and fascinating field of scientific research. An antimicrobial evaluation of the two propolis samples shows that it exerts a bactericidal activity specifically against Gram positive bacteria such as Staphylococcus aureus and Streptococcus agalactiae and a remarkable fungicidal effect against Aspergillus niger and moderately interesting against Candida famata which were estimated according to the diameters of the inhibition zones, Gram negative bacteria such as Escherichia coli, Klebsiella pneumonia and Pseudomonas aeruginosa are resistant to this antimicrobial agent (Propolis). This assessment was accompanied by a biochemical study to quantify their levels of phenolic compounds (Flavonoids) by spectrophotometry and thin layer chromatography (TLC). This study allowed us to identify the presence of flavones, flavonones and flavonols in the propolis of Eastern Algeria whose concentrations depend on its origin. High Performance Liquid Chromatography (H.P.L.C.) revealed four polyphenols, namely: Gallic Acid, Caffeic Acid, Quercetin and Catechin. This composition gave us a better classification and standardization of this product for a possible therapeutic use. According to the results we obtained, we can deduce that the propolis of El Mechrouha has a slightly higher antimicrobial activity compared to that of Ouled Driss. It also results that propolis is an attractive, interesting, important and very vast therapeutic product. In fact, propolis is a natural substance which does not need any chemical process, apart from its extraction. By the means of this work, we expect to have made our modest contribution to the valorisation of a precious product of the hive, and to have succeeded in making available to the human being a natural and efficient product. To develop the extraction and purification of these phenolic compounds so that they can be used in combination with drugs and administered at the clinical level.