Bulgarian Journal of Veterinary Medicine, 2020 ONLINE FIRST ISSN 1311-1477; DOI: 10.15547/bjvm.2020-0133
Original article
ANTIBACTERIAL AND ANTIOXIDANT ACTIVITY OF ROYAL JELLY COLLECTED FROM GEOGRAPHICAL REGIONS WITH DIFFERENT CLIMATES IN THE NORTH OF IRAN
F. HAJI MOHAMMAD1, H. KOOHSARI2 & S. H. HOSSEINI GHABOOS1 1Department of Food Sciences, Azadshahr Branch, Islamic Azad University, Azadshahr, Iran; 2Department of Microbiology; Azadshahr Branch, Islamic Azad University, Azadshahr, Iran
Summary Haji Mohammad, F., H. Koohsari & S. H. Hosseini Ghaboos, 2020. Antibacterial and anti- oxidant activity of royal jelly collected from geographical regions with different climates in the north of Iran. Bulg. J. Vet. Med. (online first).
Royal jelly is one of the most important bee products. The biological activities of royal jelly can be influenced by various factors such as geographic origin, climatic conditions, vegetation. This study was carried out to evaluate the antibacterial and antioxidant activity of royal jelly samples collected from beehives from different geographical regions including mountain, coastal and plain regions in northern Iran. Antibacterial activity of royal jelly samples against ten bacteria was determined using agar well diffusion method. The MIC and MBC of royal jelly samples were determined by the broth microdilution method. Folin-Ciocâlteu reagent and reaction with DPPH were used to determine the total phenolic content and antioxidant potential of royal jelly samples, respectively. The MIC of samples ranged from 0.78 to 12.5% and MBC from 3.12 to 50%. Samples collected from mountain regions showed the highest antibacterial activity with MIC for Gram-positive bacteria from 0.78 to 1.56% and for Gram-negative bacteria: from 1.56 to 3.12%. The total phenolic content and DPPH radical scavenging activity in royal jelly samples of the mountain region was significantly higher than those from the two regions with other climates. The results of this study indicated that the climate of the geographic region of sampling location had an effect on the antibacterial and antioxidant activity of royal jelly which may be due to differences in plant vegetation and the origin of the flowers of bees. Key words: antibacterial activity, antioxidant activity, geographical region, royal jelly
INTRODUCTION
Today, due to population growth and the tance and unwanted side effects of chemi- limitations of modern medicine, the ne- cal agents. cessity of research into introducing new The honeybee is one of the most useful antimicrobial agents of natural origin is and most developed insects. Honeybee, on strongly needed to reduce antibiotic resis- the one hand, performs pollination of Antibacterial and antioxidant activity of royal jelly collected from geographical regions with … crops, gardens, and pasture plants increas- pounds. Royal jelly composition varies ing the crop yields and vegetation residues greatly according to the season, weather in rangelands, and on the other hand pro- conditions, bee nutrition, and climatic duces honey. Besides honey, other pro- conditions of the geographical region (Sa- ducts such as royal jelly, propolis, pollen, batini et al., 2009; Fratini et al., 2016). wax have many benefits to humans. Carbohydrates make up about 30% of Royal jelly is one of the most impor- the dry weight of the royal jelly. The most tant bee products, also called King Ang- abundant sugars include fructose, glucose, bin and King Jelly, which is attributed to and sucrose. Other oligosaccharides such numerous biological activities, including as maltose, trehalose, melibiose, ribose, antibacterial and antioxidant ones. Royal and other sugars are also found in small jelly is a result of the secretion of the hy- amounts (Fratini et al., 2016). popharyngeal glands of the bees, pro- Proteins are up to 50% of the dry duced at the age of 6 to 16 days. As age weight of the royal jelly. The most impor- increases and activity changes, the secre- tant royal jelly protein compositions in- tion of these glands changes as well. All clude the family of major royal jelly pro- larvae are fed this substance in the first teins (MRJPs), antimicrobial peptides three days and after 3 days, those who (AMPs) including royalisin, apismin, become worker bees are fed honey and jelleines, royalactin, apolipophorin III-like pollen, and those to be converted into a protein, and glucose oxidase (Fratini et queen are fed only royal jelly. Royal jelly al., 2016). plays an important role in the growth of Lipids make up 3 to 19% of royal infants, completing the special sexual jelly’s dry weight. Almost 90% is made traits and the long life of the queen. Di- up of fatty acids, and the rest is from neu- gestive and milky glands in the Honey- tral fats, steroids, hydrocarbons, and phe- bees by the production of royal jelly plays nols. The most important fatty acid in the an important role in the evolution of the structure of royal jelly is the 10 hydroxy-2 hive larvae, especially the queen. It seems decanoic acid (10-HDA) which is an un- that this kind of food causes the funda- saturated fatty acid involved in the anti- mental differences between the working bacterial activity of the product. This fatty bees and the queen, so that the life of the acid is the most important marker of royal queen is more than fifty-two times the jelly quality. Also, octanoic acid at length of life of the worker bees, and in amounts lower than those of 10-HDA has term of size the queen is 23 times larger nutritional roles (Fratini et al., 2016). than the workers (Fujita et al., 2013). Various studies have been reported the In terms of physical properties, royal differences and diversity of antibacterial jelly is a concentrated and milky sub- activities of royal jelly samples collected stance with a pungent smell and specific from different geographical regions (Gar- sour taste (pH =3.44.5), with a density of cia et al., 2010; 2013; Garcia-Amoedo & 1.1 g/cm3, slightly soluble in water (Fratini de Almeida-Muradian, 2007). et al., 2016; Sadredini et al., 2016). The antioxidant activity of royal jelly Royal jelly contains 6070% water, can be attributed to phenolic compounds 1123% carbohydrates, 918% protein, phenolic acid, cinnamic acid, coumarin, 48% lipids, and 0.83% vitamins, min- isocoumarin, naphthoquinone, xanthan, eral salts and low molecular weight com- anthraquinone, flavonoids, and lignin.
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Numerous studies have pointed to the Iran including mountain, coastal, and diversity of phenolic compounds and anti- plain. Until the experiments samples were oxidant activity of royal jelly samples kept at 18 oC in the freezer of the Micro- collected from geographical regions with biology Laboratory at Islamic Azad Uni- different climates in different parts of the versity of Azadshahr branch. Samples world (Nabas et al., 2014; Ceksteryte et from beehives in the mountain region of al., 2016; Balkanska et al., 2017; Ozkok the Shahkuh-e Sofla in Gorgan township & Silici, 2017; Balkanska, 2018). The (mountain), coastal regions of the Faraha- difference in the amount of phenolic com- bad in Sari township (coastal), and plain pounds in the royal jelly depends on fac- regions of the Daland in Ramian township tors such as the weather, the herbal source (plain) were collected. The geographical used by the bee, the season, the genetic and vegetation characteristics of the re- factors, environmental factors such as the gions are presented in Table 1. type of soil (Perez-Perez et al., 2013; Sa- Serial dilution of royal jelly samples dredini et al., 2016). was carried out in sterilised distilled water The present study aimed to investigate and dilutions were prepared, resulting in the antibacterial and antioxidant effects of final concentrations of 100%, 50%, 25%, royal jelly collected from beehives from 12.5%, 6.3%, 3.12%, 1.56%, 0.78% and three geographical regions with different 0.39% v/v of each of the royal jelly sam- climates in the north of Iran. ples. Bacterial strains MATERIALS AND METHODS In this study, the antibacterial activity of Royal jelly samples royal jelly samples was evaluated against 4 bacterial native isolates and 6 bacterial Royal jelly samples were collected from reference strains. The reference strains beehives in three geographical regions used in this study comprised three Gram- with a different climate in the north of Table 1. Characteristics of royal jelly samples and collection regions Sample name and collection Climatic, geographical and vegetation characteristics region Shahkuh-e Sofla village is 65 km south-east of Gorgan in Golestan province. The village is 2540 meters above sea level with an average annual rainfall of 584 mm. Shahkuh-e It is a mountainous region with cold climate and snowy winters. The area’s vege- Sofla tation includes broad-leaved trees, shrubs and bush. Its plant species are yew, (mountain) aras, juniper, pyro, maze, beech, hornbeam, sloe, barberry, lily, blackbird, and herbaceous and bush species of oxtongue, alfalfa, sage, and artemisia. Farahabad is a coastal town 25 km north of Sari in Mazandaran Province. It is 26 Farahabad meters below sea level. The climate in this region is temperate with abundant (coastal) annual rainfall. The vegetation of the region includes agricultural lands paddy and gardens contains citrus fruits such as oranges, tangerine, and orange. Daland city of Ramian environs is located 60 km east of Gorgan in Golestan Daland province. The city is 65 meters above sea level and has fertile plains. The climate (plain) in this region is temperate. The vegetation of the region is covered with oak, free, maple and evergreen trees, and agricultural lands.
BJVM, ××, No × 3 Antibacterial and antioxidant activity of royal jelly collected from geographical regions with … negative bacteria included Escherichia Agar well diffusion method coli (PTCC 1338), Pseudomonas aerugi- In the well method, the uniform spread nosa (PTCC 1811) and Shigella dysente- culture was prepared from 0.5 McFarland riae (Persian Type Culture Collection, bacterial suspensions (1.5 × 108 CFU/mL) PTCC 1188) and three Gram-positive from all reference strains and native iso- species included Staphylococcus aureus lates on Muller Hinton Agar medium (PTCC 1112), Bacillus cereus (PTCC (Himedia, India) using sterile cotton 1154) and Enterococcus faecalis (PTCC swabs. Then, using a sterilised cork borer, 1778) which were provided from the Ira- wells with a diameter of 4.5 mm were nian Research Organization for Science created on the medium and 100 μL of and Technology (IROST) in a lyophilised each of the serial dilutions of royal jelly form. The reference strains were reco- samples was poured into wells. The plates vered in the Brain Heart Infusion (BHI) were incubated at 37 °C for 24 hours. Af- medium (Merck, Germany) for 24 h at 37 ter incubation, the diameter of the inhibi- °C. tion zone was measured using a millimeter Native isolates included S. aureus iso- ruler and recorded (Eshraghi et al., 2003; lated from the nasal cavity of the carrier, Weinstein et al., 2018). E. coli isolated from the stool, P. aerugi- nosa isolated from water, and B. cereus Determination of MIC and MBC isolated from the soil. The isolates were The determination of MIC of royal jelly identified using routine microbial labora- samples was carried out based on turbidi- tory tests for each (selective media cul- metric assay and by using the microdilu- ture, Gram stain, catalase and oxidase tion tube method in sterile 96 well mi- tests, biochemical tests). croplate. For this purpose, serial dilutions The 24-hour cultures of bacteria were of royal jelly samples were prepared in inoculated into Nutrient Broth culture nutrient broth (Merck, Germany) into the medium (Merck, Germany) and incubated wells of microplate. A suspension equiva- at 37 °C to obtain turbidity equal to 0.5 lent to 5×105 CFU/mL from each of bacte- McFarland = 1.5×108 CFU/mL. When ria was added to each of these wells and turbidity of the tube was equal to 0.5 incubated for 24 h at 37 °C. There were McFarland standard, the absorbance value also negative control tubes (serial dilu- on 625 nm wavelength was between 0.08 tions of royal jelly prepared in nutrient to 0.1 (Weinstein et al., 2018). broth without bacterial suspension) and Evaluation of the antibacterial activity of positive control tubes (bacterial suspen- royal jelly sion of 5×105 CFU/mL without royal jelly). The results for visible microbial The antibacterial activity of royal jelly turbidity were recorded after 24 h of incu- samples was carried out based on agar bation. The last dilution (lowest concen- diffusion and well method. Minimum in- tration) in which microbial turbidity was hibitory concentration (MIC) and mi- not observed, was considered as MIC. For nimum bactericidal concentration (MBC) the determination of MBC, the tube that of each specimen were tested by broth contained royal jelly with concentration microdilution tube method. higher than the MIC was cultured onto the nutrient agar medium (Merck, Germany). The MBC was defined as the lowest con-
4 BJVM, ××, No × F. Haji Mohammad, H. Koohsari & S. H. Hosseini Ghaboos centration at which no colony was ob- this wavelength and the change of the served on the agar (Weinstein et al., colour to yellow. One hundred μL of 1000 2018). ppm methanolic royal jelly solution and 100 μL methanolic DPPH∙ solution Determination of the total phenolic (2 mM) were mixed and placed in a dark content of royal jelly place. The absorbance was measured at The total phenolic content (TPC) of royal 517 nm after 15 min of incubation at jelly samples was analysed by using Folin- 25 °C. The control test was done with Ciocâlteu reagent. In this method, 0.5 mL methanol instead of royal jelly against royal jelly solution in distilled water (0.1 methanol as blank,. The experiments were g/mL) was mixed with 2.5 mL of Folin- performed in triplicate and the percent Ciocâlteu reagent (2N) and incubated for inhibition of DPPH free radicals was cal- 5 minutes. Then 2 mL of sodium carbon- culated by the equation: ate solution (75 g/L) was added into the DPPH scavenging activity (%) = royal jelly solution and incubated for 2 1 [(A – (A / A )]× 100 hours at 25 °C. After incubation for reac- Control Sample Control tion and formation of blue colour, the ab- Statistical analysis sorbance of the solution was measured at 765 nm by using a UV-Visible spectro- All data were expressed as mean±standard photometer (Jenway-UK). (Folin-Ciocâl- deviation (n=3). The data were analysed teu and from gallic acid within the con- statistically with one-way ANOVA using centration range from 0.1 to 0.9 mg/mL SPSS version 18.0 software. Comparison (R2=0.9998, y=0.1254x0.1424). The of the means with Duncan’s multi-domain total phenolic content was reported as the test was performed at a level of signifi- mean value of triplicate assays and ex- cance P≤0.05. pressed as mg gallic acid equivalent per 100 g of royal jelly sample (mgGAE/100 RESULTS g) (Singleton et al., 1999; Ozkok & Silici, 2017). Antibacterial activity of royal jelly Evaluation of the free radical Results of the mean diameter of inhibition scavenging activity zone of 10 tested bacteria against royal The free radical scavenging activity of jelly collected from beehives from three royal jelly samples was determined using different geographical regions including the 2,2-diphenyl-1-picrylhydrazyl hydrate mountain (Shahkuh-e Sofla), coastal radical (DPPH) (Brand-Williams et al., (Farahabad), and plain (Daland) regions 1995; Balkanska et al., 2017). DPPH is a are presented in Table 2. free radical, hydrophilic, stable, and pur- Royal jelly samples collected from the ple that due to the presence of single elec- beehives in the Shahkuh-e Sofla (moun- trons has a maximum absorption at 515 tain) and Farahabad (coastal) areas 517 nm. The transfer of hydrogen atoms showed significant antibacterial activity to DPPH radicals from reductive com- and in concentrations of 100 and 50% pounds such as phenols and their conver- against all tested bacteria were effective sion to non-radical forms results in a de- (P<0.05). Among these, the antibacterial crease in absorption of DPPH solution at activity of royal jelly of mountain area
BJVM, ××, No × 5 Antibacterial and antioxidant activity of royal jelly collected from geographical regions with …
Table 2. Inhibition zone diameters (mm) in the presence of different concentrations of royal jelly samples, well method (Mean±SD)
Bacteria (reference Royal jelly concentration (%) strain/native isolate) 12.5 25 50 100 Shahkuh-e Sofla (mountain) E. coli (N) 5±0.5Bb 9.5±0.5Ad 10.5±0.7Ae E. coli (R) 5.5±0Be 7.5±0.5Af B. cereus (N) 10±1Bd 14.5±1Ad B. cereus (R) 13.5±0.7Bc 16.5±0.6Ac P. aeruginosa (N) 5.5±0Ag P. aeruginosa (R) 10±0.5Ad 10.5±1.2Ae S. aureus (N) 8.5±1Ba 21.5±1.5Aa 23.5±0.9Aa S. aureus (R) 9±1Ba 17±0.8Ab 18.5±0.4Ab E. faecalis (R) 11±1Bd 13.5±0.1Ad S. dysenteriae (R) 8.5±0.5Af Farahabad (coastal) E. coli (N) 11.5±1.5Ac 12±0.5Ab E. coli (R) 5±0.5Bd 6.5±0.5Ad B. cereus (N) 10.5±0.5Ac 11±1Ab B. cereus (R) 13±1.2Ab 14.5±0.7Aa P. aeruginosa (N) 6.5±0Ad 7±0Ad P. aeruginosa (R) 5.5±0Ad 6.5±0Ad S. aureus (N) 7±0Bd 9.5±0.9Ac S. aureus (R) 14.5±0.9Aa 15.5±0.5Aa E. faecalis (R) 5.5±0Ad S. dysenteriae (R) 5±0Ad 6±0.5Ad Daland (plain) E. coli (N) 5±0Ab E. coli (R) 5±0.5Ab B. cereus (N) 5.5±0.5Ab B. cereus (R) 6±0.5Aa 7±1Aa P. aeruginosa (N) 5.5±0.9Aa 6±0.5Aa P. aeruginosa (R) 5.5±0Aa 6±1Aa S. aureus (N) 5.5±0.7Ab S. aureus (R) 6±0.5Aa E. faecalis (R) 6±1Aa 6.5±0.5Aa S. dysenteriae (R) 6.5±1Aa Diameters are given in (mm) including well (4.5 mm); N: native isolate; R: reference strain; (): no zone of inhibition. Different lowercase letters in each column indicate significant difference at the 5% probability level; different uppercase letters in each row indicate significant difference at the 5% probability level. against native isolate of S. aureus was centrations of 100, 50 and 25%, respec- interesting with mean inhibition zone di- tively (Table 2). ameter of 23.5, 21.5, and 8.5 mm, in con-
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Royal jelly samples collected from Statistical analysis of inhibition zone beehive in the plain area (Daland) showed diameters showed that S. aureus (refe- lower antibacterial activity compared to rence strain and native isolate) and B. other samples (P<0.05). This royal jelly cereus (reference strain) were the most sample with mean inhibition zone diame- sensitive to different concentrations of ters of 7, 6.5, and 6.5 mm in pure concen- royal jelly and Gram-negative bacteria E. tration, was effective against reference coli (reference strain), P. aeruginosa (na- strains of B. cereus, E., faecalis, and S. tive isolate) and S. dysenteriae were the dysenteriae respectively (Table 2). most resistant to different concentrations In general, in comparison with the of royal jelly samples (P<0.05). mean diameter of the inhibition zone of MIC and MBC of royal jelly the tested bacteria, royal jelly samples collected from beehives in the mountain Results of the MIC and MBC of royal (Shahkuh-e Sofla) and plain (Daland) jelly samples collected from beehives of regions showed the highest and lowest three geographical regions with different diameter of inhibition zone, respectively climates including mountain (Shahkuh-e (P<0.05). Therefore, the geographic re- Sofla), coastal (Farahabad), and plain gion climate of samples collecting site (Daland) regions are presented in Table 3. was found to influence significantly the MIC values of royal jelly samples were antibacterial activity of royal jelly samples within the range from 0.78 to 12.5%, and from the mountain, coastal, and plain sites MBC from 3.12 to 50% (Table 3). In (P<0.05). this method, like the well method, the As can be seen from the results of Ta- royal jelly sample collected from the ble 2, Gram-positive bacteria were sig- mountain region (Shahkuh-e Sofla) nificantly more sensitive to different con- showed the highest antibacterial activity centrations of royal jelly samples than against the tested bacteria compared with Gram-negative bacteria (P<0.05). the other two regions. MICs of royal jelly sample collected from mountain region
Table 3. Minimum inhibitory concentration (MIC, %) and minimum bactericidal concentration (MBC, %) of royal jelly samples
Shahkuh-e Sofla Farahabad Daland Bacteria (reference (mountain) (coastal) (plain) strain/native isolate) MIC MBC MIC MBC MIC MBC E. coli (N) 3.12 12.5 6.25 25 12.5 50 E. coli (R) 1.56 12.5 3.12 25 6.25 25 B. cereus (N) 0.78 3.12 3.12 6.25 6.25 12.5 B. cereus (R) 0.78 3.12 1.56 3.12 3.12 12.5 P. aeruginosa (N) 3.12 12.5 12.5 50 12.5 50 P. aeruginosa (R) 3.12 12.5 6.25 25 6.25 50 S. aureus (N) 1.56 12.5 3.12 12.5 3.12 12.5 S. aureus (R) 0.78 6.25 1.56 12.5 3.12 12.5 E. faecalis (R) 1.56 12.5 3.12 12.5 3.12 12.5 S. dysentriae (R) 3.12 12.5 6.25 25 6.25 25
N: native isolate; R: reference strain.
BJVM, ××, No × 7 Antibacterial and antioxidant activity of royal jelly collected from geographical regions with …
Table 4. Total phenolic content (TPC) and free radical scavenging activity of royal jelly samples
Total phenolic content Free radical scavenging Sample (mg GAE/100 g) activity (%) Shahkuh-e Sofla (mountain) 10.10 ± 1.35a 7.35 ± 1.05a Farahabad (coastal) 7.81 ± 0.15b 5.40 ± 0.08b Daland (plain) 5.56 ± 0.95c 3.80 ± 0.11c Different lowercase alphabet letters in each column indicate significant difference at the 5% proba- bility level. (Shahkuh-e Sofla) for Gram-positive B. of DPPH in royal jelly samples collected cereus (native isolate), B. cereus (refer- from beehives from three geographical ence strain), S. aureus (reference strain), regions with different climates. The sam- S. aureus (native isolate) and E. faecalis ple collected from the beehives in the were 0.78, 0.78, 0.78, 1.56 and 1.56% mountain region (Shahkuh-e Sofla) had respectively. These values were 3.12% for the highest total phenolic content and the inhibiting the growth of Gram-negative highest free radical scavenging activity bacteria except for the native E. coli iso- compared to samples of other two cli- late (1.56%). These results indicated the mates The royal jelly sample had the significant antibacterial activity of this bee highest total phenolic content 10.1 mg product (Table 3). gallic acid per 100 g (mg GAE/100 g). On the other hand, royal jelly samples Also, this sample had the highest DPPH collected from plain region (Daland) free radical inhibition at 1000 ppm com- showed the lowest antibacterial activity pared to other samples: 7.35% (P˂0.05). against the tested bacteria. MBC of this royal jelly sample was 50% for E. coli (native isolate) and P. aeruginosa (refer- DISCUSSION ence strain and native isolate). All tested The royal jelly sample collected from the Gram-positive bacteria including native mountain region (Shahkuh-e Sofla), isolates and reference strains were killed showed the most pronounced antibacterial with a minimum concentration of 12.5% activity against the tested bacteria of this royal jelly sample (Table 3). (P<0.05). Garcia et al. (2013) have re- The Gram-positive bacteria such as S. ported difference in antibacterial activity aureus and B. cereus were more sensitive of four royal jelly samples collected from compared to Gram-negative bacteria such different geographical regions of Argen- as E. coli and P. aeruginosa with this tina, showing that the geographic origin of method, as the MBC of royal jelly sam- royal jelly collecting location was effec- ples were in the range of 3.12 to 12.5% tive in its antibacterial activity. The results for Gram-positive bacteria but within the of their research indicated that Gram- range 12.550% for Gram-negative bacte- positive bacteria were more sensitive to ria (Table 3). different concentrations of royal jelly Total phenolic content and free radical samples than Gram-negative bacteria, scavenging activity of royal jelly which is consistent with the results of our study. They also reported that Enterococ- Table 4 shows the total phenolic content cus bacteria were more resistant than and percentage of free radical scavenging other Gram-positive genera, which is one
8 BJVM, ××, No × F. Haji Mohammad, H. Koohsari & S. H. Hosseini Ghaboos of the results of the present study (Garcia peptides plays a very important role for et al., 2013). antibacterial activity, and the presence and Antibacterial activity of royal jelly can displacement of an amino acid can affect be attributed to major royal jelly proteins this antibacterial activity (Brogden, 2005; (MRJPs), royal jelly antimicrobial pep- Pandey et al., 2011; Ebenhan et al., tides (AMPs) which include royalisin, 2014). apisimine, jelleines, and royalactin, apoli- In addition to reacting with the bacte- pophorin III-like protein and glucose oxi- rial cell membrane, the antimicrobial pep- dase. In addition to proteins, 10-hydroxy- tides of royal jelly can bind to other tar- 2-decanoic acid (10-HDA) is the most gets such as DNA, RNA, and proteins important fatty acid found in royal jelly, within the cell, and prevent the synthesis which plays a key role in its antibacterial of essential constituents of bacterial cells activity (Fratini et al., 2016). such as DNA, RNA, and proteins (Li et Most protein constituents of royal jelly al., 2012; Fratini et al., 2016). belong to a family called major royal jelly Recently, another protein called apoli- proteins (MRJPs), also called apalbumin, pophorin III, which can contribute to the that make up 8390% of royal jelly pro- antibacterial activity of royal jelly with tein constituents. In this protein family, other antimicrobial peptides has been eight proteins with molecular weights of identified (Han et al., 2011). 49 to 87 kDa have been identified (MRJP The glucose oxidase enzyme is one of 1–8). These proteins play a key role in the other proteins found in the royal jelly, feeding the queen. They can also play an which plays a role in its antibacterial ac- important role in the production of other tivity by catalysing glucose conversion to bee compounds, especially pollen grains. hydrogen peroxide (Sagona et al., 2015). In addition to these proteins, there are The most important fatty acid in the other proteins in lower amounts that have structure of the royal jelly is 10-hydroxy 2 antimicrobial activity and the antimicro- decanoic acid (10-HDA), an unsaturated bial properties of royal jelly are related to fatty acid involved in its antibacterial ac- these proteins. These antimicrobial pepti- tivity. This fatty acid is the most important des (AMPs) include royalisin, apisimine, indicator of the quality of the royal jelly. and jelleines, royalactin (Fratini et al., However, some researchers have also 2016). pointed to the antibacterial role of other The antimicrobial peptides of the royal fatty acids in royal jelly such as 3-hyd- jelly are relatively small polypeptides with roxydodecanoic acid, 11-oxododecanoic 10 to 50 amino acids, due to the presence acid, and 11-hydroxydodecanoic acid of 2 to 9 alkaline amino acid sequences (Melliou & Chinou, 2005). such as lysine, arginine, and histidine with In addition, Parveen & Rao (2012) a positive charge, whose electrostatic re- suggested that secondary metabolites in action with the surface of the cell mem- the royal jelly such as phenols, flavonoids, brane of the bacterium that has a negative glycosides, terpenoids, sterols, lignin, and charge, results in penetration of the cell saponins can show significant antibacte- membrane and, by creating holes in the rial activity against Gram-positive bacteria cell membrane of the bacterium, affects and Gram-negative bacteria. Andreas et the permeability of the membrane. In al. (2005) also found that the biological other words, the amino acid sequence of activity of the royal jelly was very diverse
BJVM, ××, No × 9 Antibacterial and antioxidant activity of royal jelly collected from geographical regions with … and presented great variations and that it agents into the bacterial cell (Nikaido, was linked to other microelements. In 2003). general, the observed difference in anti- The interesting point about comparing bacterial activity of various samples of the well method to broth microdilution royal jelly collected from different regions method for determination of MIC and with different climate is related to royal MBC in this study is that in the microdilu- jelly compounds. tion method, royal jelly samples were able Royal jelly composition could vary to inhibit the tested bacteria at very low with seasonal and regional conditions of concentrations, whereas in the well feeding (Chen & Chen, 1995; Antinelli et method antibacterial effect was observed al., 2003; Attalla et al., 2007; Sabatini et only in very high concentrations 100% al., 2009), with the type of plants from and 50%. This finding was also mentioned which nectar and pollen are supplied (Oz- in other studies (Garcia et al., 2013). The kok & Silici, 2017), with metabolites and reason for this can be the high molecular changes in the physiology of nurse bees as weight of the antimicrobial compounds of well as with the larval age (Brouwers et al., royal jelly samples, the hydrophobicity of 1987; Abd-alla et al., 1995), with bees’ the compounds, and their non-solubility in genetics and race (Liu et al., 2008; Zheng water. Given that the well method is based et al., 2011), and above all could be modi- on agar diffusion, low molecular weight fied from the storage conditions posthar- compounds can spread more and with vest (Ragab & Ibrahim, 1999; Li et al., better speed and display higher antibacte- 2008; Liu et al., 2008; Zheng et al., 2011). rial activity, while in the microdilution Several research studies correlated these method, the bacterial suspension is mixed variations of royal jelly composition to its with the desired concentration of royal antimicrobial activity (Abd-alla et al.,1995; jelly in the tube. On the other hand, many Ragab & Ibrahim, 1999; Li et al., 2008; studies attribute the antibacterial activity Liu et al., 2008; Zheng et al., 2011). of royal jelly to 10-HDA, a water- One of the results of this study was the insoluble fatty acid and one of the most higher sensitivity of Gram-positive bacte- important constituents in royal jelly that ria compared to that of Gram-negative contributes to its numerous biological bacteria against different concentrations activities (Garcia-Amoedo & Almedia- of royal jelly samples. This sensitivity is Muradian, 2003; Fratini et al., 2016). The also reported in other studies (Fujiwara et lower antibacterial activity in well method al., 2004; Garcia et al., 2010, 2013; can be related to poor propagation of this Moselhy et al., 2013). Studies have shown substance in agar. However, the direct that the cell wall of Gram-negative bacte- proximity of royal jelly with bacterial sus- ria was more resistant than that of Gram- pension in the microdilution method of positive bacteria against many antibiotics, broth solves this problem. antimicrobial chemicals, and even many The well method is used as a popular herbal drugs. This resistance can be due to and validated technique for testing anti- the lower permeability of the outer mem- bacterial activity. The disadvantage of this brane of these bacteria and the presence of method is that it is not a quantitative tech- the lipopolysaccharide layer of the cell nique. Another important point in this wall and as well as the periplasmic space technique is that hydrophobic compounds that limits the entry of antimicrobial are hardly propagated in agar. So the well
10 BJVM, ××, No × F. Haji Mohammad, H. Koohsari & S. H. Hosseini Ghaboos method is still an appropriate technique pounds in royal jelly samples depends on for determining antibacterial activity, at factors such as the climate, the origin of least as an estimate method (Kalemba & the flowers and plants used by the honey Kunicka, 2003; Wilkinson, 2006). On the bee, the season, the genetic factors and other hand, the microdilution broth environmental factors such as the type of method is a quantitative and accurate soil (Perez-Perez et al., 2013; Sadredini et method for assessing the sensitivity of al., 2016). Other reasons for these differ- microorganisms to different compounds ences are the conditions for the preserva- (Finegold & Baron, 1992). tion of royal jelly samples. They are very The biological activity of royal jelly is important for its biological properties. mainly related to active bio fatty acids, Royal jelly is extremely sensitive to light proteins, and phenolic compounds (Jam- and heat and its oxidation rapidly happens nik et al., 2012). Dezmirean et al. (2012) in direct contact with air (Buttstedt et al., analysed the total phenolic content of the 2013; Fratini et al., 2016). Pavel et al. royal jelly samples in the range of 148 to (2014) have compared commercial sam- 436 mg GAE/kg. Balkanska et al. (2017) ples with fresh and native royal jelly sam- have reported total phenolic content of 20 ples from different geographical regions samples of royal jelly from different re- of Romania and concluded that freshly gions of Bulgaria in the range of 11.66 to harvested samples had higher total pheno- 36.63 mg GAE/g while the amount of lic content, DPPH free radical inhibition, DPPH free radical inhibition of royal jelly and 10 hydroxy-2 decanoic acid content samples was reported at 11.7 to 39.39%. compared to commercial samples (Pavel Ceksteryte et al. (2016) reported total et al., 2014). The results indicated a close phenolic content of royal jelly samples relationship between antibacterial activity from Lithuania of 10.7 mg GAE/g. and antioxidant activity of royal jelly To investigate the relationship be- samples. This relationship can be attribu- tween physicochemical parameters, anti- ted to the high content of total phenolic oxidant activity and total phenolic content compounds in royal jelly which were the of 10 fresh royal jelly samples, Balkanska highest and lowest in mountain and plain (2018) reported total phenolic content of regions, respectively. the samples ranging from 11.82 to 26.07 mg GAE/g, and amount of DPPH free CONCLUSION radical inhibition of royal jelly from 15.07 to 35.59%. In another study, Ozkok & Royal jelly produced by honey bees is a Silici (2017) reported an average total natural product with great potential for phenolic content of royal jelly samples use in the food, pharmaceutical, and from different provinces of Turkey of medical industries. Samples of royal jelly 59.16 mg GAE/100 g. The total phenolic collected from the mountain region content of royal jelly samples collected showed the highest antibacterial activity from Jordan was 23.3 µg GAE/mg (Nabas against the tested bacteria with the MBC et al., 2014). As can be seen in the above in the range of 3.12 to 12.5% v/v. The studies, the difference in the total phenolic results of this study indicate that the cli- content of royal jelly samples in different mate of the geographic region of sampling parts of the world is quite evident. The location influenced the antibacterial and difference in the amount of phenolic com- antioxidant activity of the royal jelly, that
BJVM, ××, No × 11 Antibacterial and antioxidant activity of royal jelly collected from geographical regions with …
may be due to differences in plant vegeta- phenol content of royal jelly from Bul- tion and flower origin of the worker bees. garia. International Journal of Current However, study on the identification of Microbiology and Applied Sciences, 6, royal jelly compounds in different regions 578585. is recommended to identify the active Brand-Williams, W., M. F. Cuvelier & C. ingredient and its association with other Berset, 1995. Use of a free radical method environmental factors such as season, to evaluate antioxidant activity. LWT – weather, soil composition, etc. Food Science and Technology, 28, 2530. Brogden, K. A., 2005. Antimicrobial peptides: Pore formers or metabolic inhibitors in ACKNOWLEDGEMENTS bacteria? Nature Reviews Microbiology, 3, 238250. The authors thank all staff of the Microbiology Buttstedt, A., R. F. Moritz & S. Erler, 2013. laboratory of Islamic Azad University, Azad- More than royal food − major royal jelly- shahr branch. protein genes in sexuals and workers of the honeybee Apis mellifera. Frontiers in Zo- REFERENCES ology, 10, 7282. Ceksteryte, V., B. Kurtinaitiene, P. R. Vensku- Abd-alla, M. S., A. Mishref & I. M. Ghazi, tonis, A. Pukalskas, R. Kazernaviciute & J. 1995. Antimicrobial potency of royal jelly Balzekas, 2016. Evaluation of Antioxidant collected from queen cells at different lar- Activity and Flavonoid Composition in vae ages. Annals of Agricultural Sciences, Differently Preserved Bee Products. Czech 40, 597608. Journal of Food Sciences, 34, 133-142. Andreas, S., S. Peter, K. Antonius & B. Eber- Chen, I. C. & S. Y. Chen, 1995. Changes in hard, 2005. Trace and mineral elements in protein components and storage stability of royal jelly and homeostatic effects. Jour- royal jelly under various conditions. Food nal of Trace Elements in Medicine and Bi- Chemistry, 54, 195–200. ology, 19, 183189. Dezmirean, G. I., L. A. Marghitas, O. Bobis, Antinelli, J. F., S. Zeggane, R. Davico, C. D. S. Dezmirean, V. Bonta & S. Erler, Rognone, J. P. Faucon & L. Lizzani, 2003. 2012. Botanical origin cause changes in Evaluation of (E)-10-hydroxydec-2-enoic nutritional profile and antioxidant activity acid as a freshness parameter for royal of fermented products obtained from jelly. Food Chemistry, 80, 8589. honey. Journal of Agricultural and Food Attalla, K. M., A. A. Owayss & K. M. Mo- Chemistry, 60, 8028- 035. hanny, 2007. Antibacterial activities of Ebenhan, T., O. Gheysens, H. G. Kruger, J. R. beevenom, propolis, and royal jelly pro- Zeevaart & M. M. Sathekge, 2014. Antim- duced by three honey bee, Apis mellifera icrobial peptides: Their role as infection - L., hybrids reared in the same environ- selective tracers for molecular imaging. mental conditions. Annals of Agricultural BioMed Research International, 55, 1- 5. Sciences, 45, 895902. Eshraghi, S. S. & F. Seifollahi, 2003. Antibac- Balkanska, R., 2018. Correlations of physico- terial effects of royal jelly on different chemical parameters, antioxidant activity strains of bacteria. Iranian Journal of Pub- and total polyphenol content of fresh royal lic Health, 32, 2530. jelly samples. International Journal of Finegold, S. M. & E. J. Baron, 1992. Diagnos- Current Microbiology and Applied Sci- tic Micribiology, 7th edn, Editorial Medical ences, 4, 37443750. Panamericana, Bailey Scott, Buenos Aires, Balkanska, R., L. A. Marghitas & C. I. Pavel, Argentina, Capitulo, pp. 513533. 2017. Antioxidant activity and total poly-
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tion of royal jelly. Journal of ApiProduct and ApiMedical Science, 1, 16. Sadredini, A., G. H. Nehzati Paghale & F. Paper received 28.07.2020; accepted for Ghaziani, 2016. Chemical and biological publication 02.10.2020 activity of royal jelly. Iranian Journal of Bee Science and Technology, 12, 3338. Sagona, S., B. Turchi, F. Fratini, M. Giusti, B. Torracca, R. Nuvoloni, D. Cerri & A. Felicioli, 2015. Preliminary evaluation of Correspondence: glucose oxidase and its products in vitro antimicrobial activities on Paenibacillus Hadi Koohsari larvae ATCC9545 vegetative form. Bulle- Department of Microbiology, tin of Insectology, 68, 233237. Azadshahr Branch, Islamic Azad University, Singleton, V. L., R. Orthofer & R. M. Lamu- Azadshahr, Iran, ela-Raventos, 1999. Analysis of total phe- tel: (+98)17-35725925; nols and other oxidation substances and fax (+98) 17-35724003, antioxidation by means of Folin-Ciocalteu e-mail: [email protected], reagent. Methods in Enzymology, 299, http://orcid.org/0000-0002-0676-2546 152–178.
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