The alternative use of natural products, like royal jelly (RJ), may be an important tool for the treatment of infections caused by antibiotic-resistant bacteria. RJ presents a large number of bioactive substances, including antimicrobial compounds. In this study, we carried out the chemical characterization of fresh and lyophilized RJ and investigated their antibacterial effects with the purpose of evaluating if the lyophilization process maintains the chemical and antibacterial properties of RJ. Furthermore, we evaluated the antibacterial efficacy of the main fatty acid found in RJ, the 10-hydroxy-2-decenoic acid (10H2DA). Chromatographic profile of the RJ samples showed similar fingerprints and the presence of 10H2DA in both samples. Furthermore, fresh and lyophilized RJ were effective against all bacteria evaluated; that is, the lyophilization process maintains the antibacterial activity of RJ and the chemical field of 10H2DA. The fatty acid 10H2DA exhibited a good antibacterial activity against
Frequent occurrence of infections caused by bacteria resistant to antibacterial agents is a common problem in hospitals. Resistant strains of bacteria are not inhibited or killed by the antibacterial agents at concentrations of the drugs achievable in the body after normal dosage. The resistance may increase the severity of disease and drive up health care costs. Therefore, the alternative use of natural products, like bee products, may be an important tool for the treatment of these infections.
Royal jelly (RJ) is a bee product widely used in traditional Oriental medicine. It is secreted from the mandibular and hypopharyngeal glands of worker honeybees (
In its composition, RJ contains proteins (approximately 50% of its dry mass), free amino acids, vitamins, sugars, fatty acids, sterols, and minerals [
RJ also presents several pharmacological properties, such as antibacterial [
A large number of bioactive substances are present in RJ, such as antimicrobial peptides (royalisin and jelleins) [
In this study, the abbreviation 10H2DA was used in order to differentiate 10H2DA from the other fatty acid present in RJ, the 10-hydroxydecanoic acid (10HDA), which is the saturated counterpart of 10H2DA.
Fresh RJ contains approximately 66% of water. Therefore, it is perishable and must be kept refrigerated to retain its nutritional value. Besides fresh RJ, the lyophilized one also is commercially available. Lyophilization process removes the water from RJ and is carried out by means of sublimation of the water (transition directly from the solid to the gaseous state). The advantage of the lyophilized RJ is that it can be stored at room temperature. Furthermore, it is usually sold in capsules in order to facilitate its use.
In the present study, we carried out the chemical characterization of fresh and lyophilized RJ and investigated their antibacterial effects, with the purpose of evaluating if the lyophilization process maintains the chemical and antibacterial properties of RJ. Furthermore, we evaluated the antibacterial efficacy of the 10H2DA.
Fresh RJ was purchased from Apis Nativa Produtos Naturais (Araranguá, SC, Brazil). Lyophilized RJ was obtained after lyophilization process of the fresh one, using a lyophilizator (Terroni, São Carlos, SP, Brazil). 10H2DA was purchased from Chromadex (Irvine, California, USA). Methanol HPLC grade was obtained from J.T. Baker. Water was treated in Milli-Q water purification system. The following culture media were used: Mueller Hinton agar and Mueller Hinton broth, which were purchased from Difco (Detroit, MI, USA); Mueller Hinton agar with 5% sheep blood (Plast Labor, Rio de Janeiro, RJ, Brazil); and Mueller Hinton broth supplemented with 5% lysed horse blood (Ebefarma Biológica e Agropecuária, Cachoeiras de Macacu, RJ, Brazil).
Fresh and lyophilized RJ were analyzed by high-performance liquid chromatography (HPLC), using a Shimadzu apparatus equipped with a CBM-20A controller, a LC-20AT quaternary pump, a SPD-M 20A diode-array detector, and Shimadzu LC solution software, version 1.21 SP1. A Shimadzu Shim-Pack CLC-ODS (M) column (4.6 × 250 mm, particle diameter of 5
RJ was dissolved with 5 mL of methanol (HPLC grade) in 10 mL volumetric flasks, subjected to sonication for 10 min and diluted to volume with Milli-Q water. The samples were filtered through a 45
The following bacteria were used:
The broth microdilution method [
The experiments were replicated three times for each bacterium.
The data of the chemical characterization and antibacterial activity of the samples were submitted to two-way ANOVA. The data of the comparison of the bacteria were submitted to the one-way ANOVA and Bonferroni’s Multiple Comparison Test. The established significance level was 5%. Statistical analysis of data was performed using the software Graph Pad Prism 5.
The moisture contents of the lyophilized and fresh RJ were 0.96 and 69.21%, respectively. Chromatographic profile of the RJ samples showed similar fingerprints and the presence of 10H2DA in both samples (Figure
Chromatographic profile of the 10-hydroxy-2-decenoic acid (10H2DA) standard and of the lyophilized and fresh royal jelly (RJ). a: lyophilized RJ; b: fresh RJ; c: 10H2DA standard. The chromatograms were plotted at 215 nm, using HPLC, Shim-Pack CLC-ODS (M) column, and an isocratic elution with 50% methanol and 50% solution of water-phosphoric acid (0.02% v/v) over a period of 22 min at a flow-rate of 0.8 mL/min.
Content of 10-hydroxy-2-decenoic acid (10H2DA) (% w/w in relation to the dry weight) in fresh and lyophilized royal jelly.
Fresh and lyophilized RJ showed
Minimum inhibitory concentration (MIC) of lyophilized and fresh royal jelly; values are mean ± SD obtained from analyses in triplicate.
Bacterium | Royal jelly | |
---|---|---|
MIC (% w/v)a | ||
Lyophilized | Fresh | |
|
0.78 ± 0.00 | 0.78 ± 0.00 |
|
0.78 ± 0.00 | 0.78 ± 0.00 |
|
0.78 ± 0.00 | 0.78 ± 0.00 |
|
0.05 ± 0.00 | 0.05 ± 0.00 |
|
0.78 ± 0.00 | 0.78 ± 0.00 |
|
0.78 ± 0.00 | 0.78 ± 0.00 |
|
0.78 ± 0.00 | 0.78 ± 0.00 |
|
0.78 ± 0.00 | 0.78 ± 0.00 |
|
1.55 ± 0.00 | 1.55 ± 0.00 |
a: % w/v in relation to the dry weight.
The fatty acid 10H2DA was not efficacious against most of the bacteria tested (Table
Minimum inhibitory concentration (MIC) of 10-hydroxy-2-decenoic acid (10H2DA); values are mean ± SD obtained from analyses in triplicate.
Bacterium | 10H2DA |
---|---|
MIC ( |
|
|
>250 ± 0.00 |
|
>250 ± 0.00 |
|
>250 ± 0.00 |
|
62.5 ± 0.00 |
|
>250 ± 0.00 |
|
>250 ± 0.00 |
|
>250 ± 0.00 |
|
>250 ± 0.00 |
|
>250 ± 0.00 |
10H2DA is the major component of the lipid fraction of RJ; however, its content varies according to geographical origin of the sample [
The concentration of 10H2DA in fresh RJ is variable around the world and values of 0.33–2.54% were found by Genç and Aslan [
Both samples of RJ were effective against all bacteria tested. It is important to mention that the samples were not submitted to any extraction process; that is, integral RJ samples were used (fresh or lyophilized raw material). Furthermore, our findings show that the lyophilization process maintains the antibacterial activity of RJ. In an
Gram-positive (staphylococci and
Besides
Bacteria cited above also have been isolated in chronic wounds, like that present in individuals with
Melliou and Chinou [
Besides fatty acids, the antibacterial activity of RJ has been attributed to antimicrobial peptides, such as royalisin and jelleins. Royalisin is a potent antimicrobial peptide which acts against Gram-positive bacteria but not against Gram-negative bacteria [
Shen et al. [
Jelleins are effective against Gram-positive and Gram-negative bacteria [
In conclusion, fresh and lyophilized RJ maintained their 10H2DA contents and were effective against all bacteria evaluated; that is, the lyophilization process maintains the chemical and antibacterial properties of RJ. The fatty acid 10H2DA exhibited a good antibacterial activity against
The authors declare that there is no conflict of interests regarding the publication of this paper.
The authors are grateful to Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), and Financiadora de Estudos e Projetos (FINEP) for granting research fellowships and financial support.