Evaluation of the Anti-Free Radical and Antibacterial Properties of Methanolic and Aqueous Extracts of an Algerian Propolis ^†

Abstract

Propolis is a beekeeping product that has been widely known since Antiquity for its beneficial effects on human health; it has significant therapeutic potential and many biological properties. The aim of this study was to evaluate the antibacterial and antioxidant activity, using the DPPH test, of a sample of propolis from the Maadid region (in the wilaya of M’sila in eastern Algeria). The antibacterial activity of methanolic and aqueous propolis extracts was assessed in Mueller–Hinton (MH) agar medium using the disc diffusion technique on the Gram-positive microorganism Staphylococcus aureus. Our results showed that all the concentrations of the extracts tested had significant activity, with the zones of inhibition ranging from (44 ± 3.46 mm) to (10.16 ± 1.25 mm). In addition, propolis has an antioxidant power, expressed in IC50, of EAqP 0.301 ± 0.007 mg/mL and EMeth P 0.194 ± 0.016 mg/mL at a concentration of 1 mg/mL compared to the BHT control (0.087 ± 0.03 mg/mL). This study shows that the methanolic and aqueous extracts have a high level of interesting activity. We also confirm that propolis is an important natural product of the hive that can be used in a number of fields: pharmaceutical and food.

1. Introduction

Recent scientific research in the pharmaceutical and food industries has revealed that, for certain complex diseases, natural products remain a valuable source for the production of new chemical entities [1]. Propolis is a complex made by bees from their salivary secretions, wax and resinous substances. They collect it from various plant materials such as buds and young twigs [2]. Research to date has also revealed a number of biological properties of this product. Its antimicrobial activity is the main reason why it has attracted a great deal of scientific interest [3,4,5,6]. The aim of this study was to evaluate the antioxidant and antibacterial properties of the methanolic and aqueous extracts of propolis.

2. Materials and Methods

2.1. Preparation of Propolis Samples

The extraction method used was maceration with two solvents. The first solvent was methanol (80%) and the second was distilled water. Two extracts were obtained: a methanolic extract (EMeth P) and an aqueous extract (EAq P). At room temperature, the propolis was allowed to macerate by continuously stirring. The solvents were evaporated by a Rotavapor at 50 °C and stored at 4 °C until needed [7].

2.2. Assessment of Antibacterial Activity

To assess in vitro antibacterial activity against Staphylococcus aureus, the Mueller–Hinton agar disc diffusion method was used, in which the discs were impregnated with 20 μL of each extract [8].

2.3. Assessment of Antioxidant Activity

According to [9], we used a method involving DPPH⁰ (0.004%) in methanol, measuring the absorbance at 517 nm after a 30 min incubation period at room temperature.

The biological activities of the propolis were analyzed using Graph Pad Prism statistical software 8.4.3(686), with the results being expressed as mean ± SD. The Tukey test and one-way ANOVA were employed to compare the means of different variables.

3. Results

3.1. Evaluation of Antibacterial Activity

As shown in Figure 1 and Figure 2, the growth of the Gram+ bacterium Staphylococcus aureus is inhibited by the activity of the two Maadid extracts. Our results indicate that S. aureus is extremely sensitive. In comparison with the antibiotic Imipenem, there are zones of inhibition ranging from 44 to 10.16 mm (

Table 1. Diameters of inhibition zones in mm of S. aureus bacterial strain.

Extracts	Concentrations (mg/mL)	Staphylococcus aureus ATCC22923
Extract meth Propolis	200	30 ± 1.32
	400	36.13 ± 6.37
	600	44 ± 3.46
Extract aqs Propolis	200	10.83 ± 0.76
	400	11.5 ± 0.86
	600	10.16 ± 1.25

(Values are the average of three tests ± SD).

).

3.2. Evaluation of Antioxidant Activity

Our extracts differed in their ability to reduce the 2,2-diphenyl-1-picrylhydrazyl DPPH radical as a function of concentration, reaching the respective maximum values 81.15% and 76.55% (

Table 2. Percent inhibition and IC50 of methanolic and aqueous extracts of Maadid propolis.

Extracts	Percentage Inhibition (%)	IC50 (µg/mL)
Emeth P	81.15	0.194 ± 0.016 ****
Eaqx P	76.55	0.301 ± 0.007 ****
BHT (butyl hydroxytoluene)	84.85	0.087 ± 0.03 ****

(Emeth P: methanolic extract, EAqx: aqueous extract **** p ˂ 0.0001: very highly significant to compared standards).

). By comparing the two extracts studied, we noted a powerful antioxidant capacity (Figure 3). Also, the aqueous and methanolic extracts had significant antioxidant activity. The IC50 values obtained by the BHT-positive control were significantly lower.

4. Discussion

In our study, we found that propolis has extensive and important antibacterial properties against the Gram-positive bacterial strain S. aureus. Our results are similar to those of several studies that have highlighted the antibacterial effect of Algerian propolis [10,11]. Our research suggests that propolis is a great source of antioxidants. The ability of our propolis extracts to trap free radicals was confirmed through a DPPH test. The results we obtained are comparable to those of the study conducted by [4].

5. Conclusions

The antibacterial and antioxidant properties of the methanolic and aqueous extracts of propolis differ, partly because of their composition, which contains bioactive substances such as flavonoids, and their variability in polyphenol content.