International Journal of Microbiology and Mycology | IJMM |

pISSN: 2309-4796
http://www.innspub.net
Vol. 19, No. 3, p. 19-25, 2024

Open Access RESEARCH PAPER

Antimicrobial properties of selected propolis samples

Nadina A. Horril1, Santana N. Martinez1, Elmer-Rico E. Mojica*2

1
Department of Biology, Pace University, One Pace Plaza, New York, NY, USA
2
Department of Chemistry and Physical Sciences, Pace University, One Pace Plaza, New York,
NY, USA
Keywords: Propolis, Antimicrobial, Diffusion assay

Publication date: September 10, 2024

Abstract
Propolis, a natural resinous substance collected by honeybees from buds and exudates of plants, is
known for its use in the beehive as a protective barrier against intruders. It is widely used as a
popular remedy in folk medicine and as a constituent of bio-cosmetics. Recently, it is extensively
used in food and beverages to improve health and prevent diseases. Depending on the season, bee
species, vegetation and the area of collection, the chemical composition of propolis are
qualitatively and quantitatively variable, resulting in diverse biological properties. In this project,
the antimicrobial activity of several propolis samples obtained from various parts of the world
(Europe, Australia, USA and the Philippines) was determined. The propolis samples were extracted
with ethanol and the extracts were assayed with Gram-positive and Gram-negative
microorganisms. Results demonstrate that propolis is effective against a range of opportunistic
pathogens, with the samples from Russia and Washington showing the most potent antibacterial
activity, inhibiting seven microorganisms each. The propolis from the Philippines (Bicol and Laguna)
also exhibited notable antimicrobial effects, although to a slightly lesser extent.
* Corresponding Author: Elmer-Rico E. Mojica  emojica@pace.edu

19 Horril et al.
Introduction that not only have potent effects but also pose a
Propolis is a natural resinous substance collected lower risk of fostering pathogen resistance.
by honeybees from the buds and exudates of
plants. In the hive, propolis serves a crucial role In this study, we surveyed bee propolis samples
as a defensive barrier, repairing combs by sealing from different global regions to evaluate their
holes and cracks, and reinforcing the comb antimicrobial activity against both Gram-positive
borders to prevent pathogenic invasions. and Gram-negative bacteria. Through this
comparative analysis, we aim to determine which
Additionally, bees use propolis to trap intruders propolis samples exhibit the strongest
that breach the hive, further safeguarding the antimicrobial properties and to explore the
colony (Sawaya et al., 2011). underlying factors that contribute to their
effectiveness. This research could lead to the
Propolis is renowned for its wide array of discovery of new, natural antimicrobial agents
bioactive properties, including anti-inflammatory, with the potential to combat infections while
anti-tumor, cytotoxic, hepatoprotective, and mitigating the growing problem of antibiotic
immunomodulatory effects. However, its most resistance.
vital property is its ability to inhibit pathogenic
microbial invasions (Liberio et al., 2011). The Materials and methods
chemical composition of propolis is highly Bacterial cultures
variable, influenced by factors such as the There were fourteen microorganisms (seven
season, type of bee, local vegetation, and the Gram positive and seven Gram-negative) that
geographical area of collection (Lotti et al., were used in the experiment. The gram positive
2011). This diversity in composition results in bacteria used were Bacillus megaterium,
varying degrees of bioactivity among different Micrococcus luteus, Rhodococcus rhodochrous,
propolis samples. The synergistic effects of these Saphyloccus epidermidis, Sporosarcina urease,
diverse compounds make propolis particularly Staphylococcus aureus and Streptomycs griseus,
effective against microorganisms, as the while the Gram-negative bacteria used were
variability in its composition makes it difficult for Branhamella catarrhalis, Escherichia coli,
pathogens to develop resistance. Each propolis Pseudomonas aeroginosa, Pseudomonas putida,
sample presents a unique combination of Rhodospirillum rubrum, Sarcina aurantiaca and
bioactive compounds, reducing the likelihood that Serratia marcesens. These bacterial cultures were
microorganisms can adapt to and recognize the all purchased from Carolina. These cultures were
antimicrobial agents within it. inoculated into tryptic soy broth (TSB) using
sterile technique and incubated for 48 hours at
Given the substantial evidence of propolis's 37°C.
antimicrobial properties and the diversity of its
chemical makeup, a key question arises: which Propolis extracts
propolis samples are the most potent in inhibiting Propolis samples were obtained from eight
microbial growth, and what specific properties different places around world. These places are
contribute to their enhanced efficacy against Washington and California in the US, Australia,
pathogens? By analyzing propolis samples from Latvia, Russia, and the Philippines (Quezon, Bicol
various regions around the world, we can identify and Laguna). The collected propolis sample was
which areas produce the most antimicrobial-rich air-dried, weighed and macerated with absolute
propolis. This information could pave the way for ethanol (10 mL for 1 g sample) a week. The
a more targeted search for antimicrobial agents macerated extract was filtered through Whatman

20 Horril et al.
#41 filter paper to obtain the liquid extract to be The antimicrobial activity of propolis has been
used in the assay. extensively documented in the literature, with
numerous studies highlighting its effectiveness
Microbial assays against a variety of microorganisms. Previous
Autoclaved disks comprised of filter paper soaked research has demonstrated that propolis exhibits
into each individual propolis sample extract and significant antibacterial activity against
then left to air dry to remove the ethanol. Each Staphylococcus aureus (Krol et al., 1993),
microorganism was inoculated into a Petri-plate Streptococcus mutans (Koo et al., 2002),
containing tryptic soy agar (TSA) using a lazy L Streptococcus pyogenes (Bosio et al., 2000),
spreader for even bacterial distribution. Each Salmonella spp. (Orsi et al., 2005), anaerobic
autoclave disk soak with propolis extract was bacteria in the human oral cavity (Santos et al.,
placed in its appropriately labeled section on the 2002), and other pathogens, including
plate that were then incubated at 37˚C for 48 Mycobacterium species (Bankova et al., 2000).
hours. The microbial zone of inhibition around the The underlying mechanism of propolis's
soaked disks were analyzed to determine the antimicrobial activity is complex, involving at
efficacy of the propolis extract in hindering least 200 different compounds identified in
bacterial growth. The extract was not effective if various propolis samples. These compounds
any growth appeared around the disk. If the zone include fatty acids, phenolic acids and esters,
of inhibition was less than a ½ cm, it indicated flavonoids, terpenes, β-steroids, aromatic
slight bacterial inhibition by the extract. If the aldehydes and alcohols, sesquiterpenes,
zone of inhibition was greater than ½ cm, it naphthalene, and stilbene derivatives (Walker
denoted great bacterial inhibition. Ampicillin and Crane, 1987; Greenway et al., 1991;
solution was used as the positive control while Bankova et al., 2000). Among these, phenolic
autoclaved disk soaked in ethanol was used as compounds, particularly flavonoids such as
negative control. pinocembrin, galangin, and pinobanksin, have
been frequently cited as key contributors to the
Results and discussion antimicrobial activity of propolis (Krol et al.,
In the present study, the antimicrobial properties 1993; Burdock, 1998; Castaldo and Capasso,
of propolis samples from different geographical 2002).
regions, including Russia, Washington, Bicol, and
Laguna, were assessed against both Gram- Specifically, the propolis from Russia and
negative and Gram-positive bacteria. The results, Washington exhibited significant inhibition
presented in Table 1 and illustrated in Fig. 1, against seven microorganisms, surpassing the
show that propolis is effective against a wide performance of other samples. This aligns with
range of opportunistic pathogens. The data previous studies that have highlighted the
indicate that the most potent antimicrobial effects powerful antimicrobial effects of flavonoid-rich
were observed with the propolis samples from propolis, as these compounds are known to
Russia and Washington, which produced the disrupt microbial cell membranes, inhibit enzyme
largest zones of inhibition on the agar plates. activity, and interfere with bacterial metabolism
These samples demonstrated strong activity (Wang, 2000). The strong inhibition observed
against both Gram-positive and Gram-negative with these samples suggests that their flavonoid
bacteria without showing a preference for either content, particularly pinocembrin and galangin,
type, confirming the broad-spectrum nature of may be responsible for their heightened
their antimicrobial properties. antimicrobial activity.

21 Horril et al.
Fig. 1. Antimicrobial activity of propolis samples on microorganisms used in this study

The propolis sample from Bicol also showed its diverse chemical composition. This diversity in
substantial antimicrobial activity, inhibiting six activity underscores the importance of regional
different microorganisms (Table 1). However, it factors in shaping the biological properties of
exhibited only slight inhibition against four propolis, as the variation in local plant sources
additional microorganisms, suggesting a slightly directly impacts the types and concentrations of
narrower spectrum of activity compared to the bioactive compounds available to the bees.
Russian and Washington samples. This could be
attributed to variations in the chemical In terms of microbial resistance, certain bacteria,
composition of the Bicol propolis, potentially such as Serratia marcescens, Rhodococcus
reflecting differences in the local flora and rhodochrous, and Staphylococcus aurantiaca,
environmental conditions that influence the types were notably resistant to the propolis samples,
and concentrations of bioactive compounds showing minimal to no inhibition. This resistance
present. could be due to the inherent biological
characteristics of these microorganisms, such as
The propolis from Laguna demonstrated robust cell walls or efflux mechanisms that
significant inhibition against five microorganisms, reduce the efficacy of antimicrobial agents. The
with slight inhibition observed in four others. resistance observed in these strains highlights
While this sample was not as potent as those the need for further research to understand the
from Russia and Washington, it still exhibited a specific mechanisms by which certain bacteria
notable level of antimicrobial activity, reinforcing resist propolis, and whether higher
the idea that propolis from different regions concentrations or alternative formulations of
possesses unique antimicrobial properties due to propolis could overcome this resistance.

22 Horril et al.
Table 1. Antimicrobial activity of different propolis samples against Gram-positive and Gram-negative
bacteria. (Propolis sample used: 1- California; 2- Washington; 3- Australia; 4- Latvia; 5-Russia; 6-Bicol;
7-Laguna; 8- Quezon)
Microorganisms Propolis samples
1 2 3 4 5 6 7 8
Gram-positive
B. megaterium + + + + + – + –
M. luteus ++ ++ ++ ++ ++ – + +
R. rhodochrous – – – – – – – –
S. epidermidis – ++ + ++ – – – –
S. urease + ++ ++ + ++ + ++ –
S. aureus – – + – – + – +
S. griseus – ++ ++ – ++ ++ ++ ++
Gram-negative
B. catarrhalis ++ ++ ++ ++ ++ + ++ –
E. coli – ++ – – ++ – ++ ++
P. aeruginosa ++ + ++ + ++ + + -
P. putida + - + - + - + -
R. rubrum ++ ++ ++ ++ ++ ++ ++ -
S. aurantiaca - - - - - - - -
S. marcesens - - - - - - - -
++ shows significant microbial inhibition
+ shows slight microbial inhibition
– shows no microbial inhibition

Interestingly, the study found that Gram-positive preference for either Gram-negative or Gram-
bacteria were generally more susceptible to positive organisms. Despite variations in the level
inhibition by propolis compared to Gram-negative of inhibition, all samples were capable of
bacteria. This is consistent with existing inhibiting both types of bacteria to some extent.
literature, which often reports greater sensitivity This finding supports the potential of propolis as a
of Gram-positive bacteria to phenolic compounds versatile antimicrobial agent, capable of
due to their simpler cell wall structure, which addressing a wide range of bacterial infections.
lacks the outer membrane present in Gram- The broad-spectrum activity of propolis, coupled
negative bacteria (Harborn and Williams, 2000). with its ability to inhibit both Gram-positive and
Specifically, four Gram-positive bacteria - Gram-negative bacteria, highlights its potential
Streptomyces griseus, Micrococcus luteus, application in developing new antimicrobial
Sporosarcina ureae, and Staphylococcus therapies.
epidermidis - were effectively inhibited by various
propolis samples. Conversely, four Gram- These results contribute to the growing body of
negative bacteria - Rhodospirillum rubrum, evidence supporting the use of propolis as an
Branhamella catarrhalis, Escherichia coli, and effective antimicrobial agent. The study also
Pseudomonas aeruginosa -were also affected by emphasizes the importance of continuing
the propolis extracts, albeit to varying degrees. research into the diverse biological activities of
This differential susceptibility suggests that while propolis from different regions. By exploring
propolis is broadly effective, certain structural the full spectrum of microbial species affected
features of bacteria, such as the presence of an by propolis, and by analyzing the specific
outer membrane in Gram-negative bacteria, may chemical components responsible for its
influence the degree of inhibition. antimicrobial effects, researchers can better
understand how to harness the potential of
A crucial observation from this study is that no propolis in medical and environmental
single propolis sample demonstrated a clear applications.

23 Horril et al.
Moreover, the development of environmentally Burdock GA. 1998. Review of the biological
friendly antibacterial agents derived from propolis properties and toxicity of bee propolis (propolis).
presents a significant opportunity to address the Food Chem Toxicol 36, 347–363.
challenges posed by chemically synthesized
antibiotics, which contribute to environmental Castaldo S, Capasso F. 2002. Propolis, an old

degradation and the rise of antibiotic-resistant remedy used in modern medicine. Fitoterapia

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propolis, which includes compounds that act

De Castro SL, Soeiro MNC, Meirelles MNL.
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phenothiazines on the parasite and on its
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with its natural origin and multifaceted
Greenaway W, May J, Scaysbrook T, Whatley
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Conclusion
Results of this study underscore the significant
Gu Q, Zhang Y, Zhong L. 2001. Study on
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agent. The propolis samples from Russia,
Zhejiangensis 13, 161–164.
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demonstrated varying degrees of effectiveness
Harborne JB, Williams CA. 2000. Advances in
against a range of microorganisms, with the
flavonoids research since 1992. Phytochemistry
Russian and Washington samples showing
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particularly strong activity. These findings suggest
that propolis could be developed into a viable
Koo H, Rosalen PL, Cury JA, Park YK, Bowen
alternative to synthetic antimicrobial agents, WH. 2002. Effects of compounds found in
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