In recent years, fungal infections, mainly caused by Candida albicans and Rhodotorula mucilaginosa, are a major cause of morbidity and mortality, and may be life threatening, predominantly in the group of severely immunocompromised patients.

The increased use of antifungal agents also, resulted in the development of resistance to the present drugs. It makes necessary to discover new classes of antifungal compounds, to cure fungal infections. Natural products have been traditionally used in the treatment of diseases, because they are sources of many active compounds. Honey as most natural products, may have a large variance in therapeutic components depending on its origin. Thus, the floral source of honey plays an important role on its biological properties [1].

The composition of honey is rather variable and depends primarily on its floral source; however, certain external factors, such as seasonal and environmental factors and processing, also play a role. The various varieties of honey may be grouped into monofloral or multifloral [2]. The classification basically depends on whether a dominating pollen grain, originated from only one particular plant (monofloral honey) or no dominant pollen type in the sample (multifloral honey) [1,3].

The antimicrobial effect of honey is due to different substances and depends on the botanical origin of honey [4-6]. An extensive review of the antimicrobial activity of honey showed it to be derived from high sugar content, low water content, acidity, the generation of hydrogen peroxide on dilution and phytochemical components [5]. More recently, methylglyoxal was discovered to contribute to the activity of New Zealand’s manuka honey [7,8] bee defensin-1 was detected in a Dutch honey [9], and melanoidins were identified in Canadian honeys [6].

In Algeria, thanks to geographical and climatic conditions that provide a suitable environment for apiculture, honey production has been well developed. The beekeeping that has been sustained in Algeria for thousands of years, is an important agricultural activity. Recently, we reported antibacterial and antifungal properties of Algerian honeys [10-14]. The aim of this work was to evaluate the physicochemical parameters and antifungal activity characteristics of honey of different botanical sources, from three geographic origin of Algeria.

Honey samples and their Preparation

A total number of 6 honey samples were obtained from beekeepers in three regions of western Algerian: Saida (n=1); Tiaret (n=3); Relizane (n=1); Mascara (n=1) (Table 1). All samples were collected in their original packages and were transferred to the laboratory, and kept at 4-5°C, until analysis.

Pollen analysis

The preparation of honey samples for identification of botanical origin, followed the standardized method of Erdtman G [15]. Pollen identification was based on the reference collection from the LABORATOIRE DU CARI LOUVAIN-LA-NEUVE (Belgique).

Physicochemical analyses

All physicochemical tests were performed in triplicate: Water content (moisture) was determined by an Abbe-type refractometer reading at 20°C, according to the relationship between honey water content and refractive index [6,16].

Measurements of pH were performed with a pH meter ((Hanna Instruments), in a solution containing 10 g of honey in 75 ml of CO₂ free distilled water.

Honey electrical conductivity was measured at 20°C [17] with a Crison Basic 30 conductimeter. Results were expressed in microSiemens per centimeter (μS/cm) [9,18].

Ash content

Total ash was estimated by conductimetry, using the equation:

Ash content (%) = 0,083- conductivity- 0.092 [19]

Free acidity

Free acidity was determined by potentiometric titration [17]. Honey samples were homogenized in a water bath and filtered through gauze, prior to analysis. Ten grams of honey were then dissolved in 75 mL of distilled water, and alcoholic solution of phenolphthalein added. The solution was titrated with 0.1 N NaOH. Acidity (milliequivalent of acid per kg of honey) was determined, as 10 times the volume of NaOH used in titration.

Antifungal activity

Honey solutions were prepared in two concentrations: 100% and 50% (wt/vol). The samples of each honey (10 g) and sterile water were stored at 37°C for 30 min before mixing, to facilitate homogenization. The potential antifungal activity of 6 selected natural honeys, against two species of fungi was studied, using the spectrophotometric assay and agar well diffusion method.

Culture media and inoculum

Candida albicans ATCC 10231 and Rhodotorula mucilaginosa (clinical isolate), were grown on Sabouraud Dextrose Agar (SDA; Merck, Germany), for 24 h at 37°C. Yeast cells from at least five colonies (1 mm diameter) were suspended in 5 mL of sterile saline solution (0.85%), and the resulting yeast suspension was mixed for 15 s in a vortex. Then, the suspensions were adjusted by spectrophotometric method, adding saline solution, to reach the value of 0.5 in the McFarland scale corresponding to a final concentration of 3.0 ± 2.0×10⁶ cells/ mL.

Agar well diffusion method

The agar well diffusion method was employed. The honey samples were first inoculated separately on standard nutrient media with no test organisms, so as to evaluate their possible contamination. Thereafter, solidified SDA agar plates (90 mm diameter) were separately flooded with the liquid inoculums of the different test organisms, using the spread plate method. The plates were drained and allowed to dry at 37°C for 30 min, after which four equidistant wells of 8mm in diameter were punched, using a sterile cork borer at different sites on the plates. 50 μl of the different concentrations, 50% (wt/vol), and undiluted of the honey samples were separately placed in the different punched wells with 1 ml sterile syringe. The plates were allowed to stay for 15 min for pre-diffusion to take place, followed by an overnight incubation that lasted for 28 h at 37°C. The diameter of inhibition zones, including the diameter of the well, was recorded. The experiment was repeated twice.

Spectrophotometric assay

Up to 0.2 ml of the cell suspension was inoculated into 4 ml volume of honey concentration in a test tube, while inoculation of 4 ml volume of nutrient broth with 0.2 ml of the cell suspension, served as control. The optical density was determined in a spectrophotometer at 620 nm, prior to incubation (T₀) and recorded after which, the cultures were incubated for 24 hours in the dark at 37°C with constant shaking, to prevent adherence and clumping. After 24 hours of incubation, the optical densities were again determined (T₂₄) and recorded. The optical density for each replicate at T0 was subtracted from the optical density for each replicate at T₂₄. The growth inhibition for the test at each dilution was determined, using the formula: Percentage inhibition = 1-(OD test/OD control) x 100.

Where the resulting measurement recorded a negative inhibition value (growth promotion), this was reported as stimulation using the formula: Percentage inhibition = (OD test/OD control) x 100. The minimum and maximum values were 0% and 100%.

Results of honeys pollen profile analysis permits to determinate its floral origin. The identified pollens and its frequency on the six analyzed honeys are presented on Table 1. The most relevant differences among the five honeys were the type and amount of the most predominant pollen present in them.

Table1: Collection location, description and time of collection, of natural honey samples from Algeria.

The percentages of the most abundant pollen types in each honey sample, as well as the nectar and pollen character of these plants were taken into account. Following the criteria of Zander [20], when the percentage of the most abundant pollen type was over 45%, the honey sample was classified as unifloral, with an exception of eucalyptus pollen at 70%. Lower percentages were classified as polyfloral (Table 2).

Table 2: ^aFrequency classes: P – p

Physicochemical parameters

The six honey samples were studied, in terms of chemical properties, and tests were performed in triplicate. Moisture is a parameter related to the maturity degree of honey and temperature. In the present study, moisture values are between 13.76% and 16.30% (Table 3). One sample with 20.2% exceeded the limit (20%) allowed by European Community regulations, The Council of the European Union [21]. Moisture values were within the values found in Marocco honeys (between 14.64% and 18.59%) Chakir et al. [22] and less than those found in Moroccan honeys (between 14.5% and 23.6%) Terrab et al. [23]. The moisture content of honey depends on various factors such as harvesting season, degree of maturity reached in the hive and climatic factors Finola et al. [24]. Also, the moisture content of honey is highly important factor contributing to its stability against fermentation and granulation during storage [25].

Table 3: Results of physic-chemical parameters of 6 Algerian honey samples (mean ± standard deviation, n = 3).

All the Algerian honeys analysed were found to be acidic in character. Their pH values ranged from 4 to 4.6 (Table 3). In general, honey is acidic in nature, irrespective of its variable geographical origin. The pH values of Indian, Morocco, Argentinean honeys and Saudi honeys, have been found to vary between 3.7 to 4.4, 3.91 to 4.93, 3.25 to 3.32 and 3.48 to 6.06, respectively [26-28]. This parameter is of great importance, during extraction and storage of honey, as it influences the texture, stability and shelf life of honey [29].

Ash content is one of these parameters that have been associated with botanical and geographical origins of honey samples. The ash content in honey is generally small, and depends on nectar composition of predominant plants in their formation [8].

Its value in the analysed samples, ranged from 0.08 to 0.35 g% (Table 3). These results are good agreement with those of Al-Khalifa et al. [30], Sahinler and Gul [31] and Sudhanshu S et al. [32].

The electrical conductivity values of honey samples, tested in our study are listed in (Table 3).

The electrical conductivity (mS/cm) in honey samples, varied in the range of 0.20- 0.52. A linear relationship is known to exist between the ash content and the electrical conductivity, which is expressed as C = 0.14 + 1.74 A, where C is the electrical conductivity and A is the ash content Bogdanov et al. [33]. The coefficient of correlation between electrical conductivity and ash was found to be 0.98, which indicated a strong positive correlation between the two parameters. Similarly, a correlation value of 0.92 has been found to exist between the electrical conductivity and ash content, for some Algerian honeys [28]. The electrical conductivity of honey is closely related to the concentration of mineral salts, organic acids and proteins. This parameter shows great variability according to the floral origin, and is important for differentiating honeys of different floral origins.

The values of acidity obtained ranged from 8.3 meq/ kg in honey harvested, using modern method to 17.3 meq/kg (Table 3) in honey harvested using traditional method.

All honeys presented free acidity values, below 40 meq NaOH kg^-1. Free acidity may be explained by taking into account the presence of organic acids, which are proportional to the corresponding lactones, or internal esters, and some inorganic ions such as phosphate or sulphate [24]. Makhloufi et al. reported total acidity of honey from different regions of Algeria, to range from 3.0 to 22.5 meq/ kg, and this indicated the absence of undesirable fermentation. Sahinler and Gul [31] also reported varying acidity values for honey produced from sunflower, cotton, orange and pine to be 40.73, 25.24, 34.96 and 25.76 meq/ kg, respectively.

Inhibitory activity evaluation

The antifungal activity of the 6 honey samples was first measured by agar diffusion, which is suitable for a previous screening test.

The growth inhibition profiles of six honeys are presented in Figure 1 and 2. A variation in the antifungal activity with floral source was observed.

Figure 1: Antifungal activity of tested products against C. albicans ATCC 10231.

Figure 2: Antifungal activity of tested products against R. mucilaginosa.

These results are in accordance with those reported in other studies, which showed that the response of the different bacterial species to the treatment with a certain honey sample, is rather dependent on the type of honey and the individual organism within a category.

Fungal infection is becoming a serious medical problem, because of the difficulty of its control in immunocompromised individuals, and because of the emergence of multidrug-resistant fungi, although a variety of antifungal drugs have been developed [34,35]. Rhodotorula species, including R. mucilaginosa (R rubra) and R. glutinis, are often resistant to fluconazole and voriconazole [36]. The treatment of invasive Candida infections is often complicated by high toxicity, low tolerability or a narrow spectrum of activity of the current antifungal drugs, as well as an increase in the incidence of azole-resistant strains [37]. Therefore, the search for new antifungal agents is necessary and stimulates research on new chemotherapeutic agents in natural products. Different natural substances are responsible for antifungal action. Honey is a natural product that is used for its antifungal activity [14]. A number of studies have reported the antifungal properties of different types of honey. For example, Koc et al. [38] reported antifungal efficacy of various honeys, against clinical isolates of Candida albicans, C. krusei, C. glabrata and Trichosoporon spp (Figure 3). Moussa et al. [12] found that honey concentration ranging from 50% to 100%, inhibited the growth of several pathogenic microorganisms, including C. albicans and Rhodotorula sp. Nevertheless, there is no information on antifungal activity of Algeria honey against Rhodotorula mucilaginosa in the literature (Figre 4). Additionally Obaseiki-Ebor et al. [39] demonstrated the susceptibility of 72 isolates of C. albicans, to honey. Khosravi et al. [40] reported that honey had antifungal activity against Candida species such as Candida albicans, C. parapsilosis, C. tropicalis, Candida kefyr, C. glabrata, and C. dubliniensis.

Figure 3: Relationship between the level of antifungal activity against C. albicans and honey floral origin.

Previous reports demonstrated that increased honey concentrations resulted in reduced growth of C. albicans, namely 29.4% inhibition on the growth was verified, in the presence of wasbessie honey at concentrations of 25% [41].

Figure 4: Relationship between the level of antifungal activity against R. mucilogenosa and honey floral origin.

Several factors may influence the antifungal activity of honey. For example, DeMera and Angert [42] reported that honey from different phytogeographic regions, varied in their ability to inhibit the growth of yeasts, suggesting that botanical origin plays an important role in influencing the antifungal activity. In addition, there are a great variety of components, including phenolic acids, flavonoids and other biomolecules, in different honeys.

The variation in the antifungal potential of honey samples used in this study, as compared to the previous similar studies, highlights that the source of the nectars may have contributed to the difference in the antifungal activities of honey, i.e. the flowers from which bees gathered nectar to produce the honey, since flora source determines many of the attributes of honey, for example, flavor, aroma, color and composition.