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Table of Contents
BASIC RESEARCH
Year : 2018  |  Volume : 8  |  Issue : 1  |  Page : 18-22

Royal Jelly Antimicrobial Activity against Periodontopathic Bacteria


Department of Periodontics, Bapuji Dental College, Davangere, Karnataka, India

Date of Web Publication5-Mar-2018

Correspondence Address:
Sowmya Nagur Karibasappa
Bapuji Dental College, Davangere, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jid.jid_72_17

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   Abstract 


Background: Royal jelly (RJ) is a milky white highly viscous secretion from the salivary gland of the worker hive bees. RJ is a nutritive secretion that acts as biocatalyst in cell regeneration processes within the human body and exhibits antimicrobial effects. Materials and Methods: RJ antimicrobial activity against periodontopathic (American type cell culture) bacteria was investigated “in vitro.” Bacterial strains tested were Aggregatibacter actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi), and Fusobacterium nucleatum (Fn). Minimum inhibitory concentration for the strains tested was determined using the method of broth dilution with the RJ sample in serial concentrations. Results: The minimum bactericidal concentration values were identical and showed bactericidal effect in the range between 12.5 and 100 μg/ml, suggesting that periodontal pathogens tested were susceptible to RJ. Conclusion: Results showed that the inhibitory effects on Pg and Pi at different concentrations demonstrated concurrent similarity, whereas higher concentrations were necessary to inhibit the growth of Aa and Fn. However, the requirement for a standardized method for quality evaluation of RJ, i.e., qualitative, quantitative, and biological activity is a necessity, and its dosage and safety must be tested before its possible in vivo application.

Keywords: Apitherapy, minimum bactericidal concentration, minimum inhibitory concentration, periodontal pathogens, royal jelly


How to cite this article:
Coutinho D, Karibasappa SN, Mehta DS. Royal Jelly Antimicrobial Activity against Periodontopathic Bacteria. J Interdiscip Dentistry 2018;8:18-22

How to cite this URL:
Coutinho D, Karibasappa SN, Mehta DS. Royal Jelly Antimicrobial Activity against Periodontopathic Bacteria. J Interdiscip Dentistry [serial online] 2018 [cited 2018 May 25];8:18-22. Available from: http://www.jidonline.com/text.asp?2018/8/1/18/226640




   Clinical Relevance to Interdisciplinary Dentistry Top


  • The study aims at introducing a new herbal plant (royal jelly [RJ]) with medicinal value, which can combat periodontal pathogens without causing any side effects to the host body, unlike the synthetic antimicrobials
  • Minimum inhibitory concentration and minimum bactericidal concentration (MBC) procedures were carried out to assess the antibacterial activity of the jelly
  • The MBC values were identical and showed bactericidal effect in the range between 12.5 and 100 μg/ml, suggesting that periodontal pathogens tested were susceptible to RJ
  • Results showed that the inhibitory effects on Porphyromonas gingivalis and Prevotella intermedia at different concentrations demonstrated concurrent similarity whereas higher concentrations were necessary to inhibit growth of Aggregatibacter actinomycetemcomitans and Fusobacterium nucleatum
  • Dosage and safety of RJ must be tested before its possible in vivo application as a local drug delivery system in the management of chronic periodontitis.



   Introduction Top


Periodontal disease in humans has been associated with multifarious microbiota where the development of periodontitis is the result of a specific infection caused by a subgingival biofilm consisting of both anaerobic and facultative anaerobic bacteria.[1] The clinically significant cultivable periodontal bacterial species occurring at sites of periodontal disease activity are Aggregatibacter actinomycetemcomitans (Aa), Tannerella forsythia, Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi) (including the closely related Prevotella nigrescens), Fusobacterium nucleatum (Fn), and Parvimonas micra.[2],[3],[4] Treatment of periodontitis involves reduction of the total periodontal bacterial load by supragingival and subgingival mechanical debridement. However, some patients do not respond well to conventional mechanical therapy, wherein antibiotic treatment can be prescribed as adjuvants for certain patient factions.[5] Several systemic antimicrobial interventions as an adjunct to scaling and root planing have proven effective, such as metronidazole and its combination with amoxicillin.[6] On the other side, antibiotic therapy has some disadvantages such as toxicity problems, likelihood to develop allergies, resistance of anaerobic bacteria species, which pose a serious problem worldwide, and at a high cost.[7]

Consequently, new strategies for treatment of periodontal disease have been developed and especially the discovery of alternative treatments that will partially or totally exclude the use of antibiotics.

In this context, special attention has been accorded to natural medication, among which apitherapy (therapy can include the use of honey, propolis, pollen, royal jelly [RJ], and bee venom) could be a good alternative.[8]

RJ or bee's milk is a milky white highly viscous secretion from the salivary gland of the worker hive bees Apis mellifera (Apidae), which is vital for the stimulation and growth of queen bees.[9]

RJ is a nutritive secretion which contains remarkable amounts of proteins, sugars, enzymes, lipids, vitamins, hormones, mineral substances, some trace elements such as zinc, copper, and manganese which are endogenous components of the detoxifying enzymes, and some specific vital factors that act as biocatalysts in cell regeneration processes within the human body.[10] RJ has been recognized in the world market as a dietary supplement and is used in cosmetics for its alleged tonic and bio-stimulating property. It exhibits antimicrobial effects, suppresses allergic reactions, lowers the amount of blood cholesterol, prevents cell damage in cancer and HIV patients, and also aids in wound healing and growth acceleration.[9] Antimicrobial action of RJ has already been shown, and various studies report on antibacterial and antifungal actions.[9],[10],[11],[12],[13],[14],[15],[16] Minimum inhibitory concentration (MIC) is the lowest concentration of antimicrobial agent that will inhibit visible growth of bacteria after overnight incubation. Minimum bactericidal concentration (MBC) is the lowest concentration of antimicrobial agent that will prevent the growth of bacteria after subculturing on to antibiotic-free media. The closer the MIC is to the MBC, the more bactericidal the compound.[17]

However, extensive literature research procured no studies showing antimicrobial effect of RJ on periodontal pathogens; hence, the aim of this study was to investigate the antimicrobial in vitro action of RJ against most common periodontal pathogens such as Aa, Pg, Pi, and Fn.


   Materials and Methods Top


The RJ sample used in this study was obtained from Royal health foods Pte. Ltd., Singapore.

Susceptibility tests were performed using the following American type cell culture strains: Aa (29523), Pg (33277), Pi (25611), and Fn (25586) were used.

Broth dilution assays: The minimum inhibitory concentration (MIC) and the MBC of the RJ samples were determined for each of the above-mentioned bacterial strains.

Minimum inhibitory concentration

Nine dilutions of the sample were done with brain heart infusion (BHI) broth for MIC. In the initial tube, 20 μL of the sample was added into the 380 μL of BHI broth. For dilutions, 200 μL of BHI broth was added into the next nine tubes separately. Then, from the initial tube, 200 μL was transferred to the first tube containing 200 μL of BHI broth. This was considered as 10−1 dilution.

From 10−1 diluted tube, 200 μL was transferred to second tube to make 10−2 dilution. The serial dilution was repeated up to 10−9 dilution for each sample. From the maintained stock cultures of required organisms, 5 μL was taken and added into 2 mL of BHI broth. In each serially diluted tube, 200 μL of above culture suspension was added. The tubes were incubated for 24 h and observed for turbidity[17] [Figure 1].
Figure 1: Minimum inhibitory concentration procedure performed using serial dilution method

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Minimum bactericidal concentration

MIC test was done and results were noted. The tubes which were showing sensitivity, dilutions were taken, and they were inoculated on blood agar. Plates were inoculated in an anaerobic jar for 48–72 h and colonies were tallied [Figure 2].
Figure 2: Minimum bactericidal cooncentration

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   Results Top


The RJ showed clear inhibitory effects against a variety of bacteria and in similar concentrations demonstrated different inhibitory effects on different strains [Table 1]. The inhibitory effects on Pg and Pi at different concentrations demonstrated concurrent similarity, whereas higher concentrations were necessary to inhibit the growth of Aa and Fn. The MBC values were identical and showed a bactericidal effect in the range between 12.5 and 100 μg/mL [Table 2].
Table 1: Minimum inhibitory concentration of royal jelly sample obtained for each strain tested

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Table 2: Minimum bactericidal concentration

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   Discussion Top


The results of the study are indicative of the antibacterial effect on various types of periodontal pathogens. Protein and peptides in RJ partake in the defensive mechanism of honeybee against microbial pathogens through direct inactivation of microorganisms occurring in honeybee products including through induction of cytokines which aid in the regulation of transcription of defensive proteins and peptides. No complex-modifying moieties or rare amino acids are expressed, providing a broad-spectrum antibiosis against bacteria and fungi.[10]

The RJ also contains small amounts of minor proteins, the peptide antibiotics. The peptide RJ has formerly shown total inhibition of bacterial growth for Staphylococcus aureus, Listeria Monocytogenes, and  Salmonella More Details typhimurium at very high concentrations (≥200 μg/ml).[10] However, in the present study, RJ showed inhibitory activity against Pg and Pi at lower concentrations (≤100 μg/ml). Furthermore, Pi and Pg showed notable sensitivity to RJ at all concentrations [Table 1]. Royalisin, an antibiotic polypeptide, has been previously isolated from the RJ of A. mellifera. It was found to have potent antibacterial activity against Gram-positive bacteria at low concentrations but not against Gram-negative bacteria. In our study, RJ demonstrated antibacterial effect against all four periodontal pathogens. Royalisin has been involved in a defense system active against bacterial invasion of the honeybee. Bilikova et al. reported that royalisin fractions in the concentration of 180 μg/mL showed a clearly weaker inhibition against Bacillus subtilis as tetracyclin in the concentration of 50 μg/mL. In addition, the antifungal activity of a royalisin fragment isolated with dialysis membranes against the fungus Botrytis cinerea was described. Using agar diffusion tests, the antibacterial activity for this royalisin fragment against Gram-positive bacteria such as B. subtilis, Sarcina lutea, and Paenibacillus larvae strains were described. Conversely tested Gram-negative strains such as  Escherichia More Details coli and Serratia marcescens were not inhibited.[10]

Another element, the Jelleines, is the peptides with antimicrobial activity of RJ. Jelleines I, II, and III presented select antimicrobial activities against yeast and the Gram-negative and Gram-positive bacteria, whereas Jelleine IV was not active in any of the assays. They presented no similarity with the other antimicrobial peptides from the honeybees.[10]

An additional constituent, 10-Hydroxy-Delta decenoic acid (10 HDA), the major component of the lipid fraction of RJ occupies 10% of the total weight of RJ. Earlier studies showing the antimicrobial effect and minimum dosage of 10-HDA for various pathogenic microbes were E. coli 0.625 mg/mL, B. subtilis 1.25 mg/mL, and S. aureus 2.5 mg/mL, which showed that 10-HDA could effectively inhibit the growth of bacterium. Recent studies have shown that 10-HDA promotes the growth of T-lymphocyte subsets and interleukin-2, suggesting that it possesses immunoregulation and anticancer activities.[10]

Both methicillin-sensitive (MS) and methicillin-resistant (MR) S. aureus bacteria usually isolated from skin wounds have proven to be the most sensitive strains of RJ samples tested. Between them, MS was found to be the most sensitive strain taking into account that its growth was inhibited by RJ sample diluted up to a 20% w/v. The results of the inhibition tests obtained with S. aureus (MR) may be important in view of this species remaining a serious problem in the treatment of infected wounds in humans and animals. Thus, the discovery of natural products such as RJ, propolis, honey, and plant extracts, which could be used alone or with regular antibiotics, is remarkable for the reason that the efficacy of antibiotics might be increased while minimizing the probability of growth of resistant strains.[14] Considering the low production yields and high costs of RJ, these options require further investigation.

RJ mixed with starch enhances its efficacy owing to a synergistic effect created. Lower concentrations of RJ sample were necessary to inhibit the growth of Staphylococcus epidermidis while the growth of Micrococcus luteus and Enterococcus faecalis showed a remarkable sensitivity to the presence of RJ samples.[14] These results suggest that RJ contains active antimicrobial components, i.e., royalisin and 10 HDA, which are not present in other bee products.

Furthermore, recent animal studies have shown that RJ has weak estrogenic effects and prevents osteoporosis in ovariectomized rats. Like osteoporosis periodontal diseases are characteristically accompanied by bone resorption, and the positive association between osteoporosis and periodontitis has been suggested.[18],[19],[20] The studies of RJ against a wide variety of microorganisms all over the world show strong antimicrobial activity. However, none of the investigations were related to periodontal bacteria (e.g., Aa, Pi, Pg, and Fn strains) and thus the present investigation is unique wherein MIC microdilution method and MBC are used for determining antimicrobial activity of RJ against the same.

MIC and MBC are significant diagnostic aids in confirming the resistance of microbes to antibacterial drugs and also to monitor the efficacy of newer antimicrobials. Bacterial species express varying MICs and MBCs with different antimicrobials. Sensitive strains will have relatively lower values in comparison with resistant strains.[17] The stipulated concentration of antimicrobial drug in the blood should exceed the MIC by a factor of 2–8 times to offset the tissue barriers that restrict access to the infected site.[21] In the present study, the MIC and MBC values of RJ obtained for Pg and Pi were found to be lower, but for Fn and Aa, it was found to be relatively higher [Table 1] and [Table 2].

The present in vitro antibacterial assessment study is focused on an interventional approach designed to conduct a clinical trial for detecting the beneficial effects of RJ on patients at risk for periodontitis. But in vitro values of MIC and MBC may not hold good for in vivo studies due to their inherent limitations. Growth of microorganisms in vitro is exponential whereas the growth in vivo can be very slow to none.[22] So far, MIC and MBC do not indicate the true activity of the drug at the locus of infection, other than in vitro MIC and MBC, serving as substitute markers attempting to quantify the drug activity.[23]


   Conclusion Top


Our findings suggest that the RJ samples were active against the growth of the periodontal pathogens tested. The differences observed in MIC and MBC values may be related to components of RJ associated with their geographical provenance or may be linked with genetic variability between colonies. Sensitivity of bacteria to bee products varies considerably within the product and the varieties of the same product. The methods applied for antimicrobial activity of RJ are very different and therefore not comparable: beginning with the raw material preparation, the solvents choice, and also method parameters. The requirement for a standardized method for quality evaluation of RJ, i.e., qualitative, quantitative, and biological activity is a necessity. These facts guide us to persist in the near future with studies on the chemical composition of RJ and its relationship with the biological activity. Furthermore, dosage and safety of RJ must be tested before its possible in vivo application.

Acknowledgment

We would like to extend our appreciation toward Dr. Kishore G. Bhat, Head of the Department of Microbiology, Maratha Mandal Institute of Dental Sciences, Belgaum, for his technical assistance provided in support of the study.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

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Eshraghi S, Seifollahi F. Antibacterial effects of royal jelly on different strains of bacteria. Iran J Publ Health 2003;32:25-303.  Back to cited text no. 9
    
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Boukraa L. Additive activity of royal jelly and honey against Pseudomonas aeruginosa. Altern Med Rev 2008;13:330-3.  Back to cited text no. 11
    
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Eshraghi S. An evaluation of the potent inhibitory effects of royal jelly fractions against Streptomyces bacteria. Pak J Med Sci 2005;21:63-8.  Back to cited text no. 12
    
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Fujiwara S, Imai J, Fujiwara M, Yaeshima T, Kawashima T, Kobayashi K, et al. Apotent antibacterial protein in royal jelly. Purification and determination of the primary structure of royalisin. J Biol Chem 1990;265:11333-7.  Back to cited text no. 13
    
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García MC, Finola MS, Marioli JM. Antibacterial activity of royal jelly against bacteria capable of infecting cutaneous wounds. J ApiProd ApiMed Sci 2010;2:93-9.  Back to cited text no. 14
    
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Koç AN, Silici S, Kasap F, Hörmet-Oz HT, Mavus-Buldu H, Ercal BD, et al. Antifungal activity of the honeybee products against Candida spp. and Trichosporon spp. J Med Food 2011;14:128-34.  Back to cited text no. 15
    
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Shen L, Liu D, Li M, Jin F, Din M, Parnell LD, et al. Mechanism of action of recombinant acc-royalisin from royal jelly of Asian honeybee against gram-positive bacteria. PLoS One 2012;7:e47194.  Back to cited text no. 16
    
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Hidaka S, Okamoto Y, Uchiyama S, Nakatsuma A, Hashimoto K, Ohnishi ST, et al. Royal jelly prevents osteoporosis in rats: Beneficial effects in ovariectomy model and in bone tissue culture model. Evid Based Complement Alternat Med 2006;3:339-48.  Back to cited text no. 18
    
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